9-Deazapurines as Broad-Spectrum Inhibitors of the ABC Transport Proteins P-Glycoprotein, Multidrug Resistance-Associated Protein 1, and Breast Cancer Resistance Protein

Article abstract of DOI:10.1021/acs.jmedchem.7b00788

P-Glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are the three major ABC transport proteins conferring resistance to many structurally diverse anticancer agents, lead

Full text of DOI:10.1021/acs.jmedchem.7b00788

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Article
9-Deazapurines as Broad-spectrum Inhibitors of the ABC Transport Proteins
P-glycoprotein (P-gp, ABCB1), Multidrug Resistance-associated Protein
1
(MRP1, ABCC1), and Breast Cancer Resistance Protein (BCRP, ABCG2)
Katja Stefan, Sven Marcel Schmitt, and Michael Wiese
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00788 • Publication Date (Web): 10 Oct 2017
Downloaded from http://pubs.acs.org on October 11, 2017
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-Deazapurines as Broad-spectrum Inhibitors of the ABC Transport Proteins P-
glycoprotein (P-gp, ABCB1), Multidrug Resistance-associated Protein 1 (MRP1,
ABCC1), and Breast Cancer Resistance Protein (BCRP, ABCG2)
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†
†
Katja Stefan , Sven Marcel Schmitt , Michael Wiese*
University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn,
Germany
*
E-mail: mwiese@uni-bonn.de, phone: +49/228/73-5212
†
these authors contributed equally
Key words: ABC transporter; P-gp (ABCB1); MRP1 (ABCC1); BCRP (ABCG2); dual /
double inhibitor; triple inhibitor; multiple inhibition.
Abstract. P-glycoprotein (P-gp, ABCB1), Multidrug Resistance-associated Protein 1
(
MRP1, ABCC1) and Breast Cancer Resistance Protein (BCRP, ABCG2) are the three
major ABC transport proteins conferring resistance to many structurally diverse
anticancer agents leading to the phenomenon called multidrug resistance (MDR).
Much effort was put on the development of clinically useful compounds to reverse
MDR. Broad-spectrum inhibitors of ABC transport proteins can be of great use in
cancers that simultaneously co-express two or three transporters. In this work we
continued our effort to generate new, potent, nontoxic and multiple effective inhibitors
of the three major ABC transporters. The best compound was active in very low
micromolar concentration range against all three transporters and restored sensitivity
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toward daunorubicin (P-gp and MRP1) and SN-38 (BCRP) in A2780/ADR (P-gp),
H69AR (MRP1) and MDCK II BCRP (BCRP) cells. Additionally, the compound is a
non-competitive inhibitor of daunorubicin (MRP1), calcein AM (P-gp) and
pheophorbide A (BCRP) transport.
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Introduction. Since the discovery of P-gp in 1976 by Juliano and Ling, the clinical
importance of ABC transport proteins in cancer therapy has been increasingly
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recognized. These transporters are frequently expressed in several types of human
cancers,2,3,4 often simultaneously. Due to active efflux of antineoplastic agents driven
by ATP hydrolysis, their intracellular concentration is decreased, causing a lack of
response during chemotherapy called multidrug resistance (MDR). Furthermore, two
major impacts in cancer research were the discovery of MRP1 (1992 by Cole et al.),5
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and BCRP (1998 by Doyle et al.), as these transport proteins have high affinity to
structurally diverse anticancer drugs,7,8 similar to P-gp.9
The development of potent and selective inhibitors of one of the three transport
proteins was in focus of medicinal chemistry in the last four decades. Many structurally
unrelated compounds were found to have high inhibitory power against P-gp,10
MRP1,11,12,13 and BCRP.14,15 Structure-activity relationship (SAR) studies revealed
crucial partial structures and substituents being important to inhibit the target transport
protein.16,17,18 Analysis of the inhibition type and the elucidation of the mode of action
was also in focus of several studies.19,20,21
5
As MRP1 was the second most prevalent transporter in MDR, most P-gp inhibitors
have been tested against MRP1, too.11,12 This led to some dual inhibitors, e.g. the
calcium channel blocker verapamil (1; Fig. 1),22,23 the dihydropyridine derivative
bis(pyridin-4-ylmethyl)
2,6-dimethyl-4-(3-methyl-5,6-dihydro-1,4-dithiin-2-yl)-1,4-
dihydropyridine-3,5-dicarboxylate (NIK-250; 2; Fig. 1),24,25 the immunosuppressant
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cyclosporine A26,27 or the quinoline derivative 4-(diphenylacetyl)-a-[(5-
quinolinyloxy)methyl]-1-piperazineethanol-2-butenedioate fumarate (dofequidar, MS-
209, 3; Fig. 1).28,29,30 The Ely Lilly company spent huge effort in the development of
MRP1 inhibitors, some with dual inhibitory feature,31,32,33 like the tricyclic isoxazole
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amide isoster No. 13 (4; Fig. 1; IC50 (P-gp) = 1.33 µM; IC50 (MRP1) = 0.32 µM). Xenova
Ltd. specifically developed dual inhibitors of P-gp and MRP1, like the quinazolinone
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No. 40 (5; Fig. 1; IC50 (P-gp) = 0.60 µM; IC50 (MRP1) = 0.40 µM), the pyrrolopyrimidine
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No. 47 (6; Fig. 1; IC50 (P-gp) = 0.105 µM; IC50 (MRP1) = 1.31 µM), and the indolopyrimidine
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No. 49 (7; Fig. 1; IC50 (P-gp) = 3.31 µM and IC50 (MRP1) = 0.235 µM). We recently reported
on the pyrrolopyrimidine No. 31 (8; Fig. 1; IC50 (P-gp) = 6.82 µM and IC50 (MRP1) = 0.340
µM).37
Since P-gp was the most studied transport protein, many known P-gp inhibitors were
investigated for their inhibitory power against BCRP. Several structures have been
reported,
such
as
N-[4-[2-(3,4-dihydro-6,7-dimethoxy-2(1H)-
isoquinolinyl)ethyl]phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide
GF120918)38
N-[2-[[[4-[2-(3,4-dihydro-6,7-dimethoxy-2(1H)-
(elacridar,
or
isoquinolinyl)ethyl]phenyl]amino]carbonyl]-4,5-dimethoxyphenyl]-3-
39
quinolinecarboxamide (tariquidar, XR9576). Pick et al. developed tariquidar analogs
and found dual inhibitors (No. 16, 9; Fig. 2; IC50 (P-gp) = 0.421 µM and IC50 (BCRP) = 5.06
4
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µM). Köhler et al. showed in 2015 that the tetrazolic HM30181 derivative No. 23 (10;
Fig. 2; IC50 (P-gp) = 7.73 µM and IC50 (BCRP) = 0.180 µM) has affinity toward both
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transporters. Spindler et al. reported in 2016 on the tetrahydro-β-carboline derivative
No. 44 (11; Fig. 2; IC50 (P-gp) = 2.86 µM and IC50 (BCRP) = 1.10 µM) as dual inhibitor of P-
gp and BCRP.42 Flavonoids and their precursors are also good dual inhibitors as
reported by Pick et al. (flavonoid No. 21; 12; Fig. 2; IC50 (P-gp) = 9.0 µM and IC50 (BCRP)
=
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44,45,46
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.4 µM), Gu et al. (bifendate-chalcone 8f; 13; Fig. 2),
Juvale et al. (chalcone
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No. 10; 14; Fig. 2), and Kraege and Stefan et al. (quinazoline-chalcone No. 24; 15;
Fig. 2; IC50 (P-gp) = 0.48 µM and IC50 (BCRP) = 0.60 µM).20 But also quinazolines have
been reported as dual inhibtors by several authors, e.g. Juvale et al. (quinazoline No.
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; 16; Fig. 2; IC50 (P-gp) = 5.85 µM and IC50 (BCRP) = 0.190 µM),48,49 and Krapf et al. (4-
anilino-2-pyridylquinazoline No. 39; 17; Fig. 2; IC50 (P-gp) = 0.334 µM and IC50 (BCRP)
=
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0
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.299 µM). Kraege and coworkers found in 2016 the acryloylphenylcarboxamide No.
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3 (18; Fig. 2; IC50 (P-gp) = 0.494 µM and IC50 (BCRP) = 0.573 µM)51 and the
acryloylphenylcarboxylate No. 29 (19; Fig. 2; IC50 (P-gp) = 2.16 µM and IC50 (BCRP) = 1.44
µM) as two good dual P-gp and BCRP inhibitors.52
Reports on dual MRP1 and BCRP inhibitors are rare. Häcker et al. reported on rather
selective MRP1 inhibitors, the aromatic 2-(thio)ureidocarboxylic acids, which showed
only partial effects against BCRP at concentrations above 30 µM.53 Chow et al.
reported on specific flavonoids with dual inhibitory power, e.g. (4-(2-(2-(4-(4-(4-(3-(4-
fluoro-2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenoxy)butyl)-1H-1,2,3-triazol-1-
yl)ethoxy)ethoxy)phenyl)-4H-chromen-4-one (AclOAzl; 20; Fig. 3).14,54 We recently
showed pyrrolopyrimidine derivative No. 16 (21; Fig. 3; IC50 (MRP1) = 2.87 µM and IC50
(BCRP) = 4.25 µM) as moderately good dual MRP1 and BCRP inhibitors.37
Triple inhibitors of P-gp, MRP1 and BCRP with high potency are barely known. Li et
al. showed piperine (22; Fig. 4), a Piper nigrum-derived compound to affect the
accumulation of rhodamine 123, doxorubicin and mitoxanthrone in MCF7/DOX (P-gp
55
and BCRP) and A-549/DDP (MRP1) cells. The pipecolinate derivative biricodar (VX-
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10, 23; Fig. 4), specifically designed for dual P-gp/MRP1 inhibition evaluated in
clinical trials,56,57 is nowadays considered to be a triple inhibitor of P-gp, MRP1 and
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BCRP, as stated by Mindermann et al. (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-
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-methoxy-6-(
2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-
b]indole-3-
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propanoic acid 1,1-dimethylethyl ester hydrate (24; Ko143; Fig. 4), one of the most
frequently used standard inhibitors of BCRP, was found by Weidner et al. not to be
specific as it also inhibited murine P-gp and MRP1 at high concentrations above 10
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µM. Most recent reports by Pick et al. (tariquidar analogue No. 14; 25; Fig. 4; IC50 (P-
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gp) = 3.63 µM, IC50 (MRP1) = 20.77 µM, IC50 (BCRP) = 1.25 µM), Juvale et al. (quinazoline
No. 29; 26; Fig. 4; IC50 (P-gp) = 3.49 µM, IC50 (MRP1) = 7.82 µM, IC50 (BCRP) = 0.42 µM),61
Krapf et al. (4-anilino-quinoline No. 29; 27; Fig. 4; IC50 (P-gp) = 1.42 µM, IC50 (MRP1) = 2.98
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µM, IC50 (BCRP) = 1.90 µM), and us (pyrrolopyrimidine No. 23 (28; IC50 (P-gp) = 21.7 µM,
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IC50 (MRP1) = 0.981 µM, IC50 (BCRP) = 4.68 µM ) and derivative No. 9 (29; IC50 (P-gp) = 14.0
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µM, IC50 (MRP1) = 0.790 µM, IC50 (BCRP) = 20.4 µM); both Fig. 4) are the most noteworthy
triple inhibitors.
As one can see, multiple inhibitors can be found in many different structural classes.
But broad-spectrum compounds active in low micromolar range or better are rarely
known. Pyrrolo- and indolopyrimidines are promising leads with regard to inhibition of
ABC transport proteins, and cancer research in general, as different compounds of
these substance classes were found to have antiproliferative characteristics with
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antitumor activity. Since pyrrolopyrimidines
and indolopyrimidines contain the
same 9-deazapurine partial structure, we termed these compounds as stated in the
title.
In this work new 9-deazapurines were synthesized and investigated for their inhibitory
power against P-gp, MRP1 and BCRP. The most promising triple inhibitor was
evaluated with regard to its intrinsic toxicity, sensitization of A2780/ADR (daunorubicin;
P-gp), H69AR (daunorubicin; MRP1) and MDCK II BCRP (SN-38; BCRP) cells and
inhibition type.
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Results and Discussion
Chemistry
The compounds 41-61 were prepared as stated in the literature, and the intermediates
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have already been described.35,37,63,65
Preparation of the 3-(2-(piperazine-1-yl)ethyl)-1H-indole precursor (30, Fig. 5 A) for
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compounds 41-43, 48-55, and 66 was performed as stated in the literature with minor
modifications (Scheme 1 A). 3-(2-bromoethyl)-1H-indole was added to a saturated
solution of piperazine in acetonitrile and the mixture exposed to diisopropylethylamine
for 24 h, yielding the alkylated intermediate 3-(2-(piperazine-1-yl)ethyl)-1H-indole 30.
The intermediates 31a-b to 35a-b for compounds 62-65 were prepared as reported
before.37,63 2-Methoxyaniline or 4-ethoxyaniline was added to a saturated solution of
(ethoxylmethylene)malononitrile in ethanol, yielding the malononitrile derivatives 31a-
b. The pyrrole derivatives 32a-b were obtained by exposure to ethyl bromoacetate.
Addition of dimethyl formamide dimethyl acetal gave the intermediates 33a-b. Ring
closure was performed by exposure to gaseous ammonia, yielding the lactam
derivatives 34a-b. Chlorination using phosphoryl chloride gave the precursors 35a-b.
Compounds 62-65 were prepared as described before.37,63 Scheme 1 B gives an
overview of the synthetic steps.
Preparation of the intermediates 36-40 of compounds 66-74 was performed as stated
in the literature35,36,37,63 with minor modifications. 2-Aminobenzonitrile was exposed to
excess acetic anhydride yielding the acylated derivative 36. Cyclisation to the pyrrole
analog 37 was conducted with ethyl bromoacetate. Addition of dimethyl formamide
dimethyl acetate yielded derivative 38. Deacylation and simultaneous cyclisation was
achieved by exposure to gaseous ammonia, providing compound 39. Excess
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phosphoryl chloride and reflux gave the chlorinated precursor 40. Compounds 66-74
were prepared as described before.35,36,37,63,65
All other compounds were also prepared as stated in the literature.35,37,63 The identity
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of the intermediates 30, 31a-b to 35a-b and 36-40 was confirmed by H NMR
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spectroscopy. The identity of compounds 41-74 was determined by H and C NMR
spectroscopy and their purity by LC-MS analysis.
Biological Investigation
Calcein, Daunorubicin and Pheophorbide A Assays to Investigate the Compounds for
P-gp, MRP1 and BCRP Inhibition. We classified the 9-deazapurines according to their
structure into four classes: (i) 7,8-cyclisized 9-deazapurines with nitrogen containing
heterocyclic residues at position 6 (table 1); (ii) 9-deazapurines with (2-(1H-indole-3-
yl)ethyl)piperazine-1-yl residue at position 6 and aliphatic or aliphatic-aromatic
variations at position 7 (table 2); (iii) 7,8-cyclisized 9-deazapurines with oxygen
containing heterocyclic residues at position 6 and 7 (table 3); (iv) 8,9-annulated 9-
deazapurines with variations at position 6 (table 4). Figure 6 shows the obtained results
of subclass (i). The starting compound was compound 41 (Fig. 5 A), which had already
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been described by Wang et al. This applies also for standard compound 12 (SC12,
5; Fig. 5 B), which was established as standard inhibitor of MRP1 by us before.37
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According to the publication of Wang et al. in 2004 compound 41 possessed an IC50
value of 5.82 µM against P-gp and 0.6 µM for MRP1. In comparison to 75 (Fig. 5 B),
which possessed in their work an IC50 of 9.63 µM toward P-gp and 0.69 µM against
MRP1, the activities of these two compounds can be considered to be equal. Wang et
al. decided to go on with the phenethylpiperazine structure. The reasons are simple,
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since building blocks of this kind are much cheaper and easier to purchase.
Additionally, it seemed that the phenethylpiperazine derivative was a bit more selective
than the 1H-indole derivative. With multiple inhibition toward the three major ABC
transport proteins in mind, we took compound 41 as starting structure for our continuing
work. In our test system it possessed an IC50 value of 5.52 µM toward P-gp and 0.344
µM against MRP1, very similar to the results found by Wang et al. Additionally, the
compound had also inhibitory activity against BCRP (IC50 = 3.41 µM). Compared to the
compounds mentioned in the introduction, this was already a good triple inhibitor. It
was shown before that increase of the molecular weight can be beneficial with respect
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to P-gp and MRP1 inhibition. The introducing of a cycloheptyl ring system (compound
42) at positions 7 and 8 instead of the cyclohexyl ring increased activity toward the two
former transport proteins, but erased the affinity to BCRP. This made it a rather potent
dual P-gp/MRP1 inhibitor (IC50 (P-gp) = 1.71 µM and IC50 (MRP1) = 0.289 µM, respectively).
Interestingly, the elimination of the piperazine partial structure made the resulting
compound 43 a selective inhibitor of MRP1 in submicromolar range (IC50 (MRP1) = 0.957
µM).
A pyridine partial structure with alkyl linker was shown before to be beneficial for MRP1
and P-gp inhibition, as 224,25 or 2356,57,58 show. Therefore, we substituted the 4-(2-(1H-
indole-3-yl)ethyl)piperazine-1-yl residue of compounds 41-42 by a 4-(2-(pyridin-4-
yl)ethyl)piperazine-1-yl side chain. The resulting compounds 44 and 45 had dual
inhibitory power toward MRP1 and BCRP, although the BCRP affinity was in double-
digit micromolar range. Higher lipophilicity seemed to be preferred as the latter
compound had a cycloheptyl ring system compared to the former with its cyclohexyl
ring. Removal of the ethylene linker between the nitrogen heterocycle and the
piperazine partial structure gave compound 46, which was also a good dual inhibitor
of MRP1 (IC50 = 1.02 µM) and BCRP (IC50 = 4.86 µM) with no affinity to P-gp. The
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more hydrophobic counterpart compound 47 was unfortunately not analyzable due to
solubility issues. This compound promised to have an even better affinity toward MRP1
and BCRP since this correlation of activity and lipophilicity was found by us before.37
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The compound class (ii) of this work consisted of 4-(2-(1H-indole-3-yl)ethyl)piperazine-
1-yl derivatives of 9-deazapurines with aliphatic and aliphatic-aromatic variations at
position 7. Figure 7 gives the corresponding screening data. Neither the screening nor
the detailed biological investigation of MRP1 gave a tendency for a preferred side chain
at position 7, as these compounds showed all good inhibitory activity toward this
transporter between 0.5 µM and 0.3 µM. This could be expected, as all compounds of
this work were derived from known MRP1 inhibitors. But interestingly, we observed a
correlation between side chain length and inhibitory power with respect to P-gp and
BCRP. One can see the tendency between compounds 48 and 55 (Fig. 5 C). This
could be confirmed by the activity data, which is increasing with higher lipophilicity.
Compound 52 had no affinity toward BCRP, but good inhibitory power against P-gp
and MRP1 (IC50 (P-gp) = 1.28 µM and IC50 (MRP1) = 0.328 µM, respectively). But
compounds 53-55 showed good inhibitory activity toward all the ABC transporters.
While compound 53 had noticeable differences in the activity against P-gp (IC50 = 4.48
µM), MRP1 (IC50 = 0.405 µM) and BCRP (IC50 = 4.07 µM), compounds 54 and 55 were
both active in the low micromolar range or even below. It is noteworthy that compound
54 did not reach 100% accumulation in the calcein AM assay, for which we have no
explanation until now. Since this test compound did not reach the necessary value of
75% of the maximal inhibition caused by cyclosporine A toward P-gp or 24 toward
BCRP, it was not investigated in the MDR reversal-efficacy testing. Compound 55 was
the best inhibitor of the three ABC transporters, with IC50 values of 1.46 µM (P-gp),
0.501 µM (MRP1) and 1.69 µM (BCRP). This makes it an exceptional triple inhibitor,
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and as far as we know there has not been any report of that kind. Therefore, this
compound was chosen for ongoing experiments.
As one can see in Figures 1 to 4, many known P-gp inhibitors have oxygen containing
substituents, especially methoxy groups,31,41,42,43,52 like 1 (Fig. 1). Additionally, a
piperonyl partial structure was reported to be beneficial with regard to P-gp
inhibition.44,45,46,55 Therefore we synthesized new 9-deazapurine derivatives containing
oxygen heterocycles or oxygen containing substituents (compound class (iii)). The
corresponding screening results are presented in Figure 8. The piperonyl-containing
compounds (56-59) were good MRP1 inhibitors, but poor ones of P-gp or BCRP. A
furanoyl moiety with annulated cyclohexyl (60) or cycloheptyl (61) at position 7 and 8
gave dual inhibitors of MRP1 and BCRP with rather poor inhibitory activity against the
latter transport protein. Derivatization at position 7 with a 2-methoxyphenyl (62-63) or
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4-ethoxyphenyl (64-65) scaffold gave compound 63 as only representative of a triple
inhibitor, but with very poor inhibitory power toward BCRP and P-gp. Altogether, the
herein presented oxygen derivatives gave not the expected results with regard to
inhibition of P-gp, although compound 65 showed to affect both, P-gp (IC50 = 7.29 µM)
and MRP1 (IC50 = 0.859 µM).
Finally, we synthesized 8,9-annulated 9-deazapurines with variations at position 6, as
3
6
these were presented to be very potent inhibitors of MRP1 before. This moiety has
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also similarity to known BCRP inhibitors. Herein we combined these structures with
substituents that have previously37,63 and within this work been shown to affect P-gp.
Figure 9 gives a summary of the screening data. Subclass (iv) can be divided into three
categories: (a) 9-deazapurines with nitrogen heterocycle side chain (No. 66-68); (b) 9-
deazapurines with aliphatic-aromatic variations at position 6 (No. 69-72); (c) 9-
deazapurines with oxygen heterocycle containing side chains (No. 73-74). As one can
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see already from the screening results (Fig. 9), this compound class had high affinity
toward BCRP, while these compounds were only capable to achieve Imax values
between 30% and 60% with respect to inhibition of P-gp and MRP1, respectively.
Within the first category (a), the 2-(pyridin-2-yl)ethyl residue of compound 67 is
preferred compared to the 2-(1H-indole-3-yl)ethyl residue of compound 66 with respect
to MRP1 inhibition. Vice-versa, the inhibitory activity toward the other two transport
proteins is diminished. But removal of the alkyl side chain in compound 68 gave a good
triple inhibitor with IC50 values of 5.00 µM (P-gp), 0.495 µM (MRP1) and 1.81 µM
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(BCRP). This is again a rare example of a triple inhibitor in the single-digit micromolar
range. This tendency can also be observed with respect to the second category (b).
While the inhibitory activity toward MRP1 decreases from compound 69 to 72 (similar
37
to previous observations with respect to these residues ), it increases with respect to
BCRP. Compound 69 is the best compound in this set against P-gp (IC50 = 1.11 µM),
MRP1 (IC50 = 0.405 µM), BCRP (IC50 = 0.839 µM), although one might keep in mind
that this compound like the other 8,9-annulated 9-deazapurines had a reduced Imax
value (Imax (P-gp) = 26%; Imax (MRP1) = 61%; Imax (BCRP) = 28%). Figures 10 A-C show the
concentration-effect curves of compound 69 as a representative of the 8,9-annulated
9-deazapurines in comparison to the corresponding standard inhibitors cyclosporine A
(P-gp), compound 75 (MRP1) and compound 24 (BCRP). The basic mechanism
behind this phenomenon has not been clarified until now and is subject of further
investigations.
Although there is a tenfold difference between the activities toward MRP1 (IC50 = 0.146
µM) and BCRP (IC50 = 1.10 µM), compound 72 is one rare example of a good dual
inhibitor of these transport proteins. Finally, among the oxygen heterocycle-bearing
derivatives in sub-class (iii) compound 73 was found to be a moderate triple inhibitor
in single-digit and submicromolar concentration range. The furanoyl containing
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compound 74 is again a rare example of a dual MRP1/BCRP inhibitor with IC50 values
of 0.169 µM (MRP1) and 6.06 µM (BCRP).
The effect of partial inhibition as described for compounds 53-54 (ii) or the 8,9-
annulated 9-deazapurines (e.g. compound 69 (Fig. 10)) had already been observed
and reported in the literature. The quinazoline-chalcone derivative No. 17 (76; Fig. 11)
showed a partial effect on A2780/ADR cells overexpressing P-gp.20 This was also
shown for several other compound classes.67,68 But also for MRP1 reports are
available. The natural compound cepharanthine (77; Fig. 11) for example was not able
to sensitize MRP1 overexpressing IN500 and T98G glioma cells completely toward
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vincristine, etoposide and doxorubicin. The same observations have been made for
the MRP1 inhibitor 2-(4-benzhydrylpiperazin-1-yl)ethyl 5-(4,6-dimethyl-2-oxido-1,3,2-
dioxaphosphinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)nicotinate (PAK 104P, 78; Fig.
1
1) in HL60/ADR cells69 or the widely used standard MRP1 inhibitor 3-(((3-(2-(7-
chloroquinolin-2-yl)vinyl)phenyl)((3-(dimethylamino)-3-
oxopropyl)thio)methyl)thio)propanoic acid (MK571, 79; Fig. 11) in GLC /ADR cancer
4
cells.70 Last but not least, several BCRP inhibitors were shown not to reach the
inhibition value of the standard inhibitor 24, like the tetrazolic HM30181 derivative No.
41
1
0 (80; Fig. 11) reported by Köhler et al. or the 4-anilino-quinazoline No. 4 (81; Fig.
6
2
1
1) reported by Krapf and coworkers. Even compound 24 itself is a partial inhibitor
compared to fumitremorgin C (82; Fig. 11), an Aspergillus fumigatus-isolated
compound from which standard inhibitor 24 was derived. This and several other
tariquidar analogs with partial inhibiting feature have been reported by Kühnle et al.71
The basic mechanisms behind these effects are still unknown and the objective of
further investigations.
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MTT-viability Assay to Determine Intrinsic Cytotoxicity of Broad-spectrum Inhibitor 55.
Since compound 55 showed very good inhibitory activity against P-gp, MRP1 and
BCRP, and the maximal effect values were comparably high as the corresponding
effect values of the standard inhibitors cyclosporine A, 75 and 24, respectively, we
further investigated this compound with respect to its intrinsic toxicity. As can be seen
in Figure 12 A, compound 55 had no toxic effect on H69AR, MDCK II BCRP and the
corresponding sensitive cell lines, and only a marginal effect on A2780/ADR and the
wild type cells. Table 5 gives the corresponding GI50 values, which ranged mostly in
the low double-digit concentration range. Table 5 gives also the calculated therapeutic
ratios (GI50 (compound 55 in resistant cell line) divided by the IC50 (compound 55 in resistant cell line) of all
tested cell lines with respect to compound 55. It is always at least 9 times more potent
than toxic with respect to the three tested cell lines. The corresponding graphics can
be taken from Figures 12 B-D.
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MDR Reversal Assay to Determine Capability of Compound 55 to Restore Sensitivity
of ABC Transporters. Figure 13 A to C show the obtained concentration-effect curves
of daunorubicin (P-gp and MRP1) and SN-38 (BCRP) in P-gp (A2780/ADR), MRP1
(H69AR) and BCRP (MDCK II BCRP) overexpressing cells in comparison to their
parental counterparts (A2780, H69 and MDCK II, respectively). An increase of the
concentration of compound 55 from 0.1 µM to 10 µM leads to sensitization of the cell
lines as indicated by the shift of the corresponding concentration-effect curve to the left
toward the sensitive cell line. Calculation of the resistance factor (rfnative = EC50 (cytotoxic
agent; resistant cell line) divided by the EC50 (cytotoxic agent; sensitive cell line) = rfnative) without and with
supplementation (rfcompound = EC50 (cytotoxic agent; resistant cell line + compound 55) divided by the EC50
(
cytotoxic agent; sensitive cell line) = rfcompound) of compound 55 of all cell lines is shown in table
6. While the A2780/ADR cell line is 54 times more resistant against daunorubicin than
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the sensitive cell line, the resistance factor became only 4.1 in presence of 10 µM of
compound 55. The same holds for MRP1. While the doxorubicin-selected variant was
11 times more resistant than the sensitive counterpart, 10 µM of compound 55
decreased the rf to 1.2, which equals full sensitization. With respect to MDCK II BRCP
cells, the transfected variant was 20 times more resistant than the wild type. Here, 10
µM compound 55 fully restored sensitivity, making the transfected cells as sensible
toward SN-38 as the wild type. Vice versa, the potentiation factor (pf = EC50 (cytotoxic agent;
resistant cell line) divided by EC50 (cytotoxic agent; resistant cell line + compound 55) arose with increasing
compound concentration (table 6). We also calculated the maximal sensitization (pf
divided by the rfnative), which represents the percentage sensitization normalized on the
resistance factor. While compound 55 was able to fully restore sensitivity of MRP1 and
BCRP overexpressing cells, this effect was only partially observed with regard to
A2780/ADR cells. This is caused by the resistance factor of 54, which is high compared
to the other two cell lines.
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To further evaluate the compound with respect to its sensitizing property, we plotted
the resultant GI50 values of each calculated concentration-effect curve from Figure 13
A-C against the concentration of compound 55. Minus infinity (bottom value)
corresponds to the GI50 value of the resistant cell line without compound. Infinity (top
value) corresponds to the GI50 value of the sensitive cell line. As one can see from
Figure 14, the resultant concentration-effect curves for all three tested ABC
transporters had EC50 values below 1 µM, that are presented in Table 7. This means
that only submicromolar concentrations are needed to reduce the resistance of the
tested cell lines by half.
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Determination of Mode of Inhibition. We analyzed compound 55 with respect to the
type of inhibition. As can be seen in Figure 15, in all three cases the type of inhibition
is non-competitive as indicated by the reduced transport velocity (vmax), while the
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affinity (K ) stayed the same. Taken together, compound 55 is a non-competitive
M
inhibitor of P-gp-mediated transport of calcein AM, MRP1-mediated transport of
daunorubicin, and BCRP-mediated transport of pheophorbide A.
Conclusion.
In this work we continued our effort to generate new potent inhibitors of the ABC
transport proteins P-gp, MRP1 and BCRP. While the literature gives much information
about selective and dual inhibitors with respect to these three transporters, the field of
triple inhibition is rather poorly investigated.
In this manuscript we report not only on compounds with good dual inhibitory power
toward P-gp/MRP1 (42, 52, 65) and BCRP/MRP1 (46, 72, 74), but also on compound
55 as a highly potent and efficient modulator of all three investigated transport proteins.
With respect to accumulation of the fluorescent substrates calcein (P-gp), daunorubicin
(MRP1) and pheophorbide A (BCRP), compound 55 showed to be superior in
comparison to the other tested compounds as the resulting Imax values were higher
than 75% compared to the effect-values of the corresponding standard inhibitors. The
IC50 values with respect to these three transport proteins were in the low single-digit
micromolar range (P-gp and BCRP) or even in the higher nanomolar range (MRP1).
Sensitization of the cell lines toward the cytotoxic agents daunorubicin (P-gp and
MRP1) as well as SN-38 (BCRP) determined via the MDR reversal assay showed
compound 55 to fully sensitize the latter two transporters, while P-gp overexpressing
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cells still kept partial resistance toward daunorubicin. Furthermore, we showed that
only submicromolar concentrations are needed to reduce the resistance of the
corresponding cell lines by half. Up to now there has never been any report on a
compound with this ability. The type of inhibition of the P-gp-mediated transport of
calcein AM, the MRP1-mediated transport of daunorubicin and the BCRP-mediated
transport of pheophorbide A was non-competitive in all three cases as determined by
Cornish-Bowden analysis. Due to the fact that compound 55 is at least 9 times more
potent than toxic toward A2780/ADR, H69AR and MDCK II BCRP cells, and it
sensitizes cancer cells significantly in submicromolar concentrations, it represents a
great lead structure for ongoing research in cancer chemotherapy and a good
candidate for in vivo studies.
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Experimental Section.
Chemistry. Materials. Chemicals used for synthesis were purchased from Acros
Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Applichem GmbH
(
Darmstadt, Germany), Fisher Scientific GmbH (Waltham, MA, USA), Merck Millipore
Billerica, MA, USA), Sigma-Aldrich (St. Louis, MO, USA) and VWR International
(
GmbH (Darmstadt, Germany) and used without further purification. For monitoring
reaction progress analytical thin layer chromatography was used with silica gel F254
coated aluminum plates (Merck Millipore). Methylene chloride/acetone (18:1 and 9:1,
respectively) or methylene chloride/acetone/methanol (9:1:1) was used as eluent for
all intermediates 31a-b to 35a-b and 36-40 and target compounds 41-74, except for
compound 30, which was analyzed with methylene chloride/acetone/ethanol/ammonia
solution (25%; 1:1:1:0.5). UV detection was conducted at 254 nm. The target
compounds 41-74 were purified by column chromatography with silica gel 60 (43-60
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µm, Merck Millipore) and gradient elution (petroleum ether/methylene chloride 1:1,
methylene chloride, methylene chloride/acetone 18:1, methylene chloride/acetone 9:1,
methylene chloride/acetone/methanol 18:1:1, methylene chloride/acetone/methanol
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:1:1, in each case 200 mL). Intermediate 30 was additionally eluted with 200 mL of
methylene chloride/acetone/ethanol/ammonia solution (25%; 1:1:1:0.5 or 1:1:1:1).
While the identity of intermediates 30, 31a-b to 35a-b and 36-40 was determined by
1H NMR spectroscopy, the identity and purity of the target compounds 41-74 was
1
13
confirmed by H, as well as C NMR spectroscopy and LC-MS analysis. The NMR
spectra were recorded in DMSO-d or CDCl as stated in the spectroscopic data. Using
a Bruker Avance 500 MHz (500/126 MHz) the chemical shifts (δ) are expressed as
6
3
1
13
ppm calibrated to the solvent signal of DMSO ( H NMR δ 2.50; C NMR δ 39.5) or
1
13
CDCl ( H NMR δ 7.26; C NMR δ 77.1). Distortionless enhancement by polarization
3
transfer (DEPT) and attached proton test (APT) techniques were employed to assign
13C signals. Spin multiplicities of the compounds 30-74 are depicted as singulet (s),
doublet (d), doublet of doublets (dd), triplet of doublets (td), triplet (t), doublet of triplets
(
dt), quartet (q), quintet (quint) and multiplet (m). LC-MS analysis of the target
compounds 41-74 was conducted using an Agilent 1100 series with photo diode array
DAD) detector (Agilent Technologies, Santa Clara, CA, USA) and a Nucleodur column
(
100-5 C18 (Macherey-Nagel, Düren, Germany) followed by ESI mass spectrometry
using an API 2000 Triple Quadrupole mass spectrometer (Applied Biosystems,
Waltham, MA, USA) and Sciex Analyst Software version 1.5.1. The purity of all
investigated compounds in biological testing was determined as ≥ 95% unless
otherwise stated.
General Procedure for the Preparation of Compound 30. This synthesis was performed
6
6
as described with minor modifications. 3-(2-bromoethyl)-1H-indole was dissolved in
acetonitrile and given to a saturated solution of piperazine (4 eq.) in acetonitrile.
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Addition of Hünig base (diisopropylethylamin, 6 eq.) started the reaction at room
temperature. After 24 hours, the Hünig base hydrochloride was filtered off and the clear
filtrate was evaporated, resulting in an orange oil containing the title compound.
Column chromatography performed as stated above resulted in the purified alkylated
product.
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3-(2-(piperazine-1-yl)ethyl)-1H-indole (30). Orange solid; yield: 52%. H NMR (500
MHz, DMSO-d
6
) δ 10.74 (s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.13
(d, 2.3 Hz, 1H), 7.04 (dd, J = 8.1, 7.0 Hz), 6.95 (dd, J = 7.9, 7.0, Hz), 2.85-2.81 (m,
13
2
H), 2.81-2.76 (m, 4H), 2.59-2.53 (m, 2H), 2.48-2.40 (m, 4H), 1.87 (s, 1H). C NMR
(
126 MHz, DMSO-d
6
) δ 136.3, 127.4, 122.6, 120.9, 118.4, 118.2, 112.7, 111.4, 59.5,
53.2 (2C), 45.2 (2C), 22.3.
General Procedure for the Preparation of Compounds 36a-b. The intermediates 31a-
b to 35a-b for compounds 62-65 were synthesized as described before.37,63 2-
methoxyaniline or 4-ethoxyaniline was given to a saturated solution of
(ethoxymethylene)malononitrile in ethanol. The reaction progress could be followed
visibly as the resultant aniline derivative (31a or 31b) precipitated as a yellow solid.
After 1 hour, the solid was filtered off, rinsed with ethanol and dried. 2-(((2-
methoxyphenyl)amino)methylene)malononitrile
(31a)
or
2-(((4-
ethoxyphenyl)amino)methylene)malononitrile (31b) was dissolved in dimethyl
formamide and heated to 100 °C in the presence of excess potassium carbonate. Ethyl
bromoacetate was added dropwise in an equimolar amount and heating was continued
for 5 hours. After pouring into ice-cold water, the resulting precipitate containing the
pyrrole derivatives 32a or 32b was filtered off and dried. Ethyl 3-amino-4-cyano-1-(2-
methoxyphenyl)-1H-pyrrole-2-carboxylate (32a) or ethyl 3-amino-4-cyano-1-(4-
ethoxyphenyl)-1H-pyrrole-2-carboxylate (32b) was dissolved in dimethyl formamide
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and 5 eq. of dimethyl formamide dimethyl acetale were added. After 5 hours, the
solvent was evaporated and the viscous liquid containing the pyrrole derivatives 33a
or 33b was used without further purification or characterization. The brown oil
containing ethyl 4-cyano-3-(((dimethylamino)methylene)amino)-1-(2-methoxyphenyl)-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
H-pyrrole-2-carboxylate
(33a)
or
ethyl
4-cyano-3-
(
((dimethylamino)methylene)amino)-1-(4-ethoxyphenyl)-1H-pyrrole-2-carboxylate
33b) was dissolved in ethanol and gaseous ammonia was passed in for 5 hours. The
(
solvent was evaporated and a sodium hydroxide solution (10% m/m) was added,
stirring the reaction mixture for 30 minutes. Neutralization with acetic acid gave the
cyclisation product 34a or 34b. 5-(2-methoxyphenyl)-4-oxo-4,5-dihydro-3H-
pyrrolo[3,2-d]pyrimidine-7-carbonitrile (34a) or 5-(4-ethoxyphenyl)-4-oxo-4,5-dihydro-
3H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile (34b) was dissolved in excess phosphoryl
chloride chloride and refluxed overnight. The reaction mixture was poured into ice-cold
water leading to a precipitate, which was filtered off, rinsed with fresh water and dried,
yielding
the
title
compounds
4-chloro-5-(2-methoxyphenyl)-5H-pyrrolo[3,2-
d]pyrimidine-7-carbonitrile (35a) or 4-chloro-5-(4-ethoxyphenyl)-5H-pyrrolo[3,2-
d]pyrimidine-7-carbonitrile (35b) as precursors to generate the target compounds 62-
65.
2
-(((2-Methoxyphenyl)amino)methylene)malononitrile (31a) Yellow needles; yield:
46%. This intermediate was used without further purification or characterization.
2
7
2
-(((4-Ethoxyphenyl)amino)methylene)malononitrile (31b) Yellowish powder; yield:
1
3%. H NMR (500 MHz, DMSO-d
6
) δ 10.98 (s, 1H), 8.34 (s, 1H), 7.33 (d, J = 9.1 Hz,
H), 6.91 (d, J = 9.1 Hz, 2H), 4.00 (q, J = 7.0 Hz, 1H), 1.30 (t, J = 7.0 Hz, 3H).
Ethyl 3-amino-4-cyano-1-(2-methoxyphenyl)-1H-pyrrole-2-carboxylate (32a) Brown
1
powder; quantitative yield. H NMR (500 MHz, DMSO-d
6
) δ 7.63 (s, 1H), 7.21 (d, J =
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.9 Hz, 2H), 6.93 (d, J = 9.0, 2H), 5.90 (s, 2H), 4.05 (q, J = 7.0), 3.99 (q, J = 7.1 Hz,
H), 1.33 (t, J = 7.0 Hz, 3H), 0.97 (t, J = 7.1 Hz, 3H).
Ethyl 3-amino-4-cyano-1-(4-ethoxyphenyl)-1H-pyrrole-2-carboxylate (32b) Brown
powder; yield: 92%. This intermediate was used without further purification or
characterization.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
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2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ethyl
4-cyano-3-(((dimethylamino)methylene)amino)-1-(2-methoxyphenyl)-1H-
pyrrole-2-carboxylate (33a) Brown oil; quantitative yield. This intermediate was used
without further purification or characterization.
Ethyl 4-cyano-3-(((dimethylamino)methylene)amino)-1-(4-ethoxyphenyl)-1H-pyrrole-
2-carboxylate (33b) Brown oil, yield: 94%. This intermediate was used without further
purification or characterization.
5
-(2-Methoxyphenyl)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile
34a) greywhite powder; yield: quantitative. This intermediate was used without further
purification or characterization.
(
5-(4-Ethoxyphenyl)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile (34b)
Greywhite powder; yield: quantitative. This intermediate was used without further
purification or characterization.
4-Chloro-5-(2-methoxyphenyl)-5H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile
(35a)
1
Greywhite powder; yield: not determined. H NMR (500 MHz, DMSO-d
6
) δ 8.94 (s, 1H),
8.89 (s, 1H), 7.63-7.58 (m, 2H), 7.28 (dd, J = 8.8, 1.1 Hz, 1H), 7.15 (td, J = 7.6, 1.2 Hz,
1H), 3.74 (s, 3H).
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4-Chloro-5-(4-ethoxyphenyl)-5H-pyrrolo[3,2-d]pyrimidine-7-carbonitrile
(35b)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Greywhite powder: yield: quantitative. This intermediate was used without further
purification or characterization.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure for the Preparation of Compound 40. Due to low yields when
attempting to react 2-aminobenzonitrile with ethyl bromoacetate, the primary amine
was acylated first. Therefore, 2-aminobenzonitrile was dissolved in excess acetic
anhydride and heated to 100 °C using microwave irradiation at 200 W for 2 hours. The
reaction mixture was poured into water. The resulting precipitate was rinsed with fresh
water and filtered off, resulting in the acylated compound 36. The following reactions
were performed as already published35,36,37,63 but with minor modifications. N-(2-
cyanophenyl)acetamide (36) was dissolved in dimethyl formamide and heated to 100
°C in the presence of excess potassium carbonate. An equimolar amount of ethyl
bromoacetate was added dropwise and heating was continued for 5 hours. Afterwards,
the reaction mixture was poured into ice-cold water and stirred for one hour. The
resultant precipitate was filtered off and dried, yielding the pyrrole derivative 37. A
solution of compound 37 in dimethyl formamide and 3.5 equivalents of dimethyl
formamide dimethyl acetale was heated to 100°C for 5 h. Afterwards the solvent was
evaporated resulting in a brown oil, which was used without further purification and
characterization.
This
oil
containing
ethyl
1-acetyl-3-
(
((dimethylamino)methylene)amino)-1H-indole-2-carboxylate (38) was dissolved in
ethanol and gaseous ammonia was passed through for 5 hours. Addition of a 10%
(m/m) sodium hydroxide solution followed by stirring for 30 minutes. After neutralization
with acetic acid, the precipitating solid was rinsed with fresh water and dried, resulting
in the cyclisized compound 39. The dried 3,5-dihydro-4H-pyrimido[5,4-b]indole-4-one
(
39) was dissolved in excess phosphoryl chloride and refluxed overnight. The reaction
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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5
5
5
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5
5
5
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mixture was poured into ice-cold water, yielding the chlorinated precursor 4-chloro-5H-
pyrimido[5,4-b]indole (40).
1
N-(2-Cyanophenyl)acetamide (36) White needles; yield: 70%. H NMR (500 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DMSO-d
6
) δ 10.11 (s, 1H), 7.78 (dd, J = 7.6, 1.4 Hz, 1H), (dd, J = 8.3, 7.5 Hz, 1H),
1
3
7
.57 (dd, J = 8.4, 0.9 Hz, 1H), 7.32 (td, J = 7.6, 1.2 Hz, 1H), 2.09 (s, 3H). C NMR
(126 MHz, DMSO-d
6
) δ 168.8, 140.5, 133.9, 133.3, 125.7, 125.6, 117.0, 107.4, 23.3.
1
Ethyl 3-amino-1H-indole-2-carboxylate (37) Brown powder; yield: 62%. H NMR (500
MHz, DMSO-d ) δ 8.05 (dt, J = 8.4, 0.8 Hz, 1H), 7.96 (dd, J 7.9, 1.2 Hz, 1H), 7.48 (dd,
6
J = 8.5, 7.2 Hz, 1H), 7.27 (dd, J =8.0, 7.2 Hz, 1H), 6.70 (s, 2H), 4.29 (q, J = 7.0 Hz,
13
2
H), 2.37 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSO-d ) δ 171.3,
6
162.1, 144.5, 138.5, 130.0, 122.0, 121.8, 121.2, 115.2, 104.3, 59.9, 26.5, 14.6.
Ethyl 1-acetyl-3-(((dimethylamino)methylene)amino)-1H-indole-2-carboxylate (38)
Brown oil; yield: 62%. This intermediate was used without further purification or
characterization.
1
3,5-Dihydro-4H-pyrimido[5,4-b]indole-4-one (39) greywhite powder; yield: 40%. H
NMR (500 MHz, DMSO-d
6
) δ 12.32 (s, 1H), 12.03 (s, 1H), 7.98 (s, 1H), 7.98 (dt, J 8.0,
0.9 Hz, 1H), 7.52 (dt, J = 8.3, 0.8, 1H), 7.45 (dd, J = 8.2, 6.9 Hz, 2H), 7.22 (dd, J = 7.9,
1
3
6.9 Hz, 1H). C NMR (126 MHz, DMSO-d
6
) δ 154.7, 141.5, 138.7, 138.3, 127.1, 122.7,
121.3, 120.4, 120.3, 112.9.
1
4
-Chloro-5H-pyrimido[5,4-b]indole (40) brown powder; yield: 52%. H NMR (500 MHz,
DMSO-d
6
) δ 12.33 (s, 1H), 8.84 (s, 1H), 8.25 (dt, J 8.0, 1.0 Hz 1H), 7.70 (dd, J = 7.8,
13
6
.6 Hz, 1H), 7.68 (dt, J = 8.3, 1.3 2H), 7.37 (dd, J = 8.0, 6.6 Hz, 1H). C NMR (126
MHz, DMSO-d
6
) δ 148.7, 147.0, 141.9, 141.4, 130.9, 128.2, 121.7, 121.2, 120.0, 113.1.
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The target compounds 41-61, 62-65 and 66-74 were prepared as already published by
us and others.35,36,37,63,65 A solution of chlorinated precursors, 35a-b or 40 in 0.5 mL
dimethyl formamide and 0.5 mL of piperazine precursor 30, other piperazine
derivatives or tryptamine in 0.5 mL dimethyl formamide was heated to 110 °C with 200
W microwave irradiation for 60 minutes. The resulting product was first purified by
column chromatography as stated above and recrystalyzed from ethanol and
petroleum ether (1:1).
37
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
4-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-6,7,8,9-tetrahydropyrimido[4,5-
1
b]indolizine-10-carbonitrile (41) Yellow powder; yield: 58%. H NMR (500 MHz, DMSO-
d
6
) δ 10.76 (s, 1H), 8.47 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.32 (dt, J = 8.2, 0.8 Hz, 1H),
7.15 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.1, 7.1 Hz, 1H), 6.96 (dd, J = 7.9, 7.0 Hz, 1H),
4
2
.36 (t, J = 5.6 Hz, 2H), 3.37-3.32 (m, 4H), 3.11 (t, J = 6.5 Hz, 2H), 2.89 (t, J = 7.3 Hz,
1
3
H), 2.71-2.65 (m, 6H), 1.98-1.88 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 154.4,
6
151.3, 151.0, 150.2, 136.3, 127.4, 123.6, 120.9 (2C), 118.4, 118.3, 114.5, 112.7,
111.5, 83.6, 58.9, 52.4 (2C), 50.4 (2C), 46.1, 22.9, 22.6, 22.6, 18.4. LC-MS: (m/z) calc.:
+
4
25.2; found: 426.4 [M+H] . Purity: 91%.
4-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-7,8,9,10-tetrahydro-6H-
pyrimido[4',5':4,5]pyrrolo[1,2-a]azepine-11-carbonitrile (42) Pale yellow crystals; yield:
1
9
9%. H NMR (500 MHz, DMSO-d
6
) δ 10.74 (s, 1H), 8.48 (s, 1H), 7.52 (d, J = 7.9 Hz,
1H), 7.32 (d, J = 8.1, 1H), 7.16 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.0, 7.0 Hz, 1H), 6.96
(
dd, J = 7.9, 7.1 Hz, 1H), 4.55-4.42 m, 2H), 3.41-3.30 (m, 4H), 3.09 (t, J = 5.5 Hz, 2H),
1
3
2
.89 (t, J = 7.3 Hz, 2H), 2.75-2.62 (m, 6H), 1.91-1.82 (m, 2H), 1.80-1.71 (m, 4H) C
) δ 156.6, 153.7, 151.1, 149.6, 136.2, 127.4, 122.6, 120.9,
18.4, 118.3, 117.9, 114.6, 112.6, 111.4, 85.3, 58.9, 52.2 (2C), 49.8 (2C), 47.4, 29.9,
NMR (126 MHz, DMSO-d
6
1
2
+
7.9, 27.1, 25.6, 22.5. LC-MS: (m/z) calc.: 439.3; found: 440.3 [M+H] . Purity: 99%.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
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5
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4
-((2-(1H-Indole-3-yl)ethyl)amino)-6,7,8,9-tetrahydropyrimido[4,5-b]indolizine-10-
1
carbonitrile (43) Pale yellow crystals; yield: 13%. H NMR (500 MHz, DMSO-d
6
) δ 10.80
(s, 1H), 8.27 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 2.1
Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 6.92 (t, J = 5.6 Hz, 1H), 4.32
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(t, J = 6.2 Hz, 2H), 3.75 (dd, J = 14.0, 6.0 Hz, 2H), 3.04-2.98 (m, 4H), 2.03-1.95 (m,
1
3
2H), 1.86-1.79 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 152.2, 149.8, 147.6, 147.5,
6
136.4, 127.6, 122.8, 121.1, 118.5, 118.3, 115.0, 113.9, 112.1, 111.5, 82.1, 45.6, 41.4,
+
25.0, 23.0, 22.1, 18.1. LC-MS: (m/z) calc.: 356.2; found: 357.2 [M+H] . Purity: 96%.
4-(4-(2-(Pyridin-4-yl)ethyl)piperazine-1-yl)-6,7,8,9-tetrahydropyrimido[4,5-b]indolizine-
1
10-carbonitrile (44) Yellow powder; yield: 42%. H NMR (500 MHz, DMSO-d
6
) δ 8.46
(s, 1H), 8.44 (dd, J = 4.4, 1.6 Hz, 2H), 7.28 (dd, J = 4.4, 1.6 Hz, 2H), 4.34 (t, J = 5.6
Hz, 2H), 3.38-3.31 (m, 4H), 3.11 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 7.3 Hz, 2H), 2.71-2.60
(
m, 6H), 1.98-1.87 (m, 4H). 13C NMR (126 MHz, DMSO-d
) δ 154.3, 151.2, 151.0,
6
150.2, 149.5 (2C), 136.3, 124.4 (3C), 118.2, 114.4, 83.6, 58.1, 52.6 (2C), 50.2, 46.0
+
(2C), 31.8, 22.9, 22.6, 18.4. LC-MS: (m/z) calc.: 387.2; found: 388.3 [M+H] . Purity:
9
6%.
4-(4-(2-(Pyridin-4-yl)ethyl)piperazine-1-yl)-7,8,9,10-tetrahydro-6H-
pyrimido[4',5':4,5]pyrrolo[1,2-a]azepine-11-carbonitrile (45) Yellow powder; yield: 42%.
1H NMR (500 MHz, DMSO-d
) δ 8.49 (s, 1H), 8.46 (d, J = 4.6 Hz, 2H), 7.29 (d, J = 4.7
6
Hz, 2H), 4.52-4.45 (m, 2H), 3.20-3.17 (m, 4H), 3.12 (t, J = 6.4 Hz, 2H), 2.82 (t, J = 7.3
13
Hz, 2H), 2.70-2.59 (m, 6H), 1.91-1.83 (m, 2H), 1.83-1.71 (m, 4H). C NMR (126 MHz,
DMSO-d
6
) δ 156.6, 153.7, 151.1, 149.6, 149.5, 149.5 (2C), 124.4 (3C), 117.9, 114.6,
8
4
5.3, 58.2, 52.0 (2C), 49.7 (2C), 47.4, 31.8, 29.9, 27.9, 27.1, 25.6. LC-MS: (m/z) calc.:
+
01.2; found: 402.2 [M+H] . Purity: 100%.
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4-(4-(Pyridin-2-yl)piperazine-1-yl)-6,7,8,9-tetrahydropyrimido[4,5-b]indolizine-10-
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
carbonitrile (46) Pale yellow crystals; yield: 55%. H NMR (500 MHz, DMSO-d
6
) δ 8.49
(s, 1H), 8.15-8.12 (m, 1H), 7.56 (dd, J = 8.9, 6.9 Hz, 1H), 6.88 (d, J = 8.6 Hz), 6.68 (dd,
J = 7.1, 4.9 Hz, 1H), 4.42 (t, J = 5.5 Hz, 2H), 3.67 (t, J = 5.3 Hz, 4H), 3.42 (t, J = 5.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
Hz, 4H), 3.13 (t, J = 6.4 Hz, 2H), 1.99-1.88 (m, 4H). C NMR (126 MHz, DMSO-d ) δ
6
1
59.2, 154.4, 151.3, 151.1, 150.3, 147.7, 137.8, 118.4, 114.4, 113.6, 107.5, 83.6, 50.1
(
2C), 46.1, 44.5 (2C), 23.0, 22.6, 18.4. LC-MS: (m/z) calc.: 359.2; found: 360.1 [M+H]+.
Purity: 93%.
4
-(4-(Pyridin-2-yl)piperazine-1-yl)-7,8,9,10-tetrahydro-6H-
pyrimido[4',5':4,5]pyrrolo[1,2-a]azepine-11-carbonitrile (47) Yellow crystals; yield:
1
58%. H NMR (500 MHz, DMSO-d
6
) δ 8.51 (s, 1H), 8.14 (dd, J = 4.8, 1.9 Hz, 1H), 7.57
(
(
dd, J = 8.8, 6.8 Hz, 1H), 6.90 (d, J = 8.6 Hz), 6.68 (dd, J = 7.1, 4.9 Hz, 1H), 4.57-4.50
m, 2H), 3.76-3.63 (m, 4H), 3.44-3.35 (m, 4H), 3.11 (t, J = 5.0 Hz, 2H), 1.89-1.73 (m,
+
6H). LC-MS: (m/z) calc.: 373.2; found: 374.2 [M+H] . Purity: 100%.
4
-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-methyl-5H-pyrrolo[3,2-d]pyrimidine-7-
1
carbonitrile (48) yellow crystals; yield: 29%. H NMR (500 MHz, DMSO-d
6
) δ 10.74 (s,
1H), 8.51 (s, 1H), 8.44 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.32 (dt, J = 8.1, 0.9 Hz, 1H),
7
.16 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.1, 7.0 Hz, 1H), 6.96 (dd, J = 7.9, 7.0 Hz, 1H),
1
3
4
.01 (s, 3H), 3.42-3.37 (m, 4H), 2.89 (t, J = 7.3 Hz, 2H), 2.73-2.65 (m, 6H). C NMR
) δ 154.9, 151.4, 150.1, 141.5, 136.3, 127.4, 122.6, 121.0, 118.6,
18.4, 118.3, 114.4, 112.6, 111.5, 85.4, 58.9, 52.3 (2C), 50.2 (2C), 36.8, 22.5. LC-MS:
(126 MHz, DMSO-d
6
1
+
(
m/z) calc.: 385.2; found: 386.3 [M+H] . Purity: 100%.
4-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-ethyl-5H-pyrrolo[3,2-d]pyrimidine-7-
1
carbonitrile (49) Yellow crystals; yield: 25%. H NMR (500 MHz, DMSO-d
6
) δ 10.76 (s,
1H), 8.61 (s, 1H), 8.55 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.16
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(
(
d, J = 2.2 Hz, 1H), 7.06 (dd, J = 8.0, 7.0 Hz, 1H), 6.96 (dd, J = 7.9, 7.0 Hz, 1H), 4.35
q, J = 7.2 Hz, 2H) 3.38-3.33 (m, 4H), 2.90 (t, J = 7.4 Hz, 2H), 2.76-2.66 (m, 6H), 1.38
1
3
(t, J = 7.2 Hz, 3H). C NMR (126 MHz, DMSO-d
6
) δ 155.0, 151.4, 150.6, 140.6, 136.3,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
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2
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7
8
9
0
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8
9
0
1
5
9
27.4, 122.7, 121.0, 118.4, 118.3, 117.9, 114.4, 112.6, 111.5, 86.2, 58.9, 52.4 (2C),
+
0.2 (2C), 44.1, 22.6, 16.1. LC-MS: (m/z) calc.: 399.2; found: 400.2 [M+H] . Purity:
7%.
4
-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-propyl-5H-pyrrolo[3,2-d]pyrimidine-7-
1
carbonitrile (50) White crystals; yield: 21%. H NMR (500 MHz, DMSO-d
6
) δ 10.74 (s,
1
H), 8.60 (s, 1H), 8.56 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.32 (dt, J = 8.1, 0.8 Hz, 1H),
.16 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.1, 7.0 Hz, 1H), 6.96 (dd, J = 7.9, 7.0 Hz, 1H),
7
4.27 (t, J = 7.4 Hz, 2H) 3.38-3.32 (m, 4H), 2.90 (t, J = 7.3 Hz, 2H), 2.72-2.64 (m, 6H),
13
1
.76 (p, J = 7.3 Hz, 2H), 0.72 (t, J = 7.4 Hz, 3H). C NMR 126 MHz, DMSO-d ) δ
6
1
55.1, 151.4, 150.6, 141.2, 136.3, 127.4, 122.6, 120.9, 118.4, 118.3, 118.0, 114.3,
112.6, 111.5, 85.9, 58.9, 52.5 (2C), 50.8, 50.2 (2C), 24.2, 22.5, 10.7. LC-MS: (m/z)
+
calc.: 413.2; found: 414.3 [M+H] . Purity: 91%.
4
-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-cyclopropyl-5H-pyrrolo[3,2-
1
d]pyrimidine-7-carbonitrile (51) White crystals; yield: 39%. H NMR 500 MHz, DMSO-
d
6
) δ 10.74 (s, 1H), 8.47 (s, 1H), 8.46 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.32 (d, J = 8.1,
1
H), 7.15 (d, J = 2.3 Hz, 1H), 7.04 (dd, J = 8.1, 7.1 Hz, 1H), 6.96 (dd, J = 7.9, 7.1 Hz,
1H), 3.97 (p, J = 3.6 Hz, 1H) 3.60-3.50 (m, 4H), 2.88 (t, J = 7.8 Hz, 2H), 2.70-2.62 (m,
13
6
H), 1.15-1.02 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 154.4, 151.3, 150.1, 140.2,
6
1
36.3, 127.4, 122.6, 120.9, 118.5, 118.4, 118.2, 114.4, 112.6, 111.4, 85.6, 58.9, 52.5
(
2C), 49.8 (2C), 32.1, 22.5, 8.5 (2C). LC-MS: (m/z) calc.: 411.2; found: 412.2 [M+H]+.
Purity: 96%.
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1
carbonitrile (52) Yellow crystals; yield: 11%. H NMR (500 MHz, DMSO-d
6
) δ 10.70 (s,
1H), 8.74 (s, 1H), 8.58 (s, 1H), 7.62-7.58 (m, 2H), 7.54-7.50 (m, 3H), 7.43 (d, J = 7.8
Hz, 1H), 7.30 (dt, J = 8.1, 0.8 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 8.1, 7.1
Hz, 1H), 6.94 (dd, J = 7.9, 7.0 Hz, 1H), 3.12-3.06 (m, 4H) 2.71 (t, J = 8.3 Hz, 2H), 2.44
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
(
t, J = 8.2 Hz, 2H), 2.16-2.08 (m, 4H). C NMR (126 MHz, DMSO-d
6
) δ 153.2, 151.9,
1
50.2, 141.4, 137.9, 136.3, 129.6 (2C), 128.5, 127.3, 125.3 (2C), 122.5, 120.9, 118.3,
118.2, 115.3, 114.1, 112.5, 111.4, 88.1, 58.7, 51.5 (2C), 48.8 (2C), 22.2. LC-MS: (m/z)
+
calc.: 447.2; found: 448.1 [M+H] . Purity: 93%.
4
-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-benzyl-5H-pyrrolo[3,2-d]pyrimidine-7-
1
carbonitrile (53) white crystals; yield: 18%. H NMR (500 MHz, DMSO-d
6
) δ 10.74 (s,
1
H), 8.65 (s, 1H), 8.56 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.33-7.28 (m, 3H), 7.27-7.23
(
m, 1H), 7.16-7.14 (m, 1H), 7.14-7.13 (m, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.04 (dd, J =
8.1, 7.0 Hz, 1H), 6.96 (dd, J = 7.9, 7.0 Hz, 1H), 5.54 (s, 2H), 3.39-3.33 (m, 4H) 2.90-
13
2
.85 (m, 2H), 2.71-2.61 (m, 6H). C NMR (126 MHz, DMSO-d ) δ 155.2, 151.7, 150.9,
6
1
41.7, 137.0, 136.3, 128.8 (2C), 128.1, 127.4, 127.1 (2C), 122.6, 120.9, 118.4, 118.3,
117.9, 114.00, 112.6, 111.4, 87.0, 58.3, 52.3 (2C), 52.1, 50.0 (2C), 22.5. LC-MS: (m/z)
+
calc.: 461.2; found: 462.0 [M+H] . Purity: 97%.
4
-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-phenethyl-5H-pyrrolo[3,2-
1
d]pyrimidine-7- (54) white crystals; yield: 9%. H NMR (500 MHz, DMSO-d
6
) δ 10.73
(
s, 1H), 8.55 (s, 1H), 8.50 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.33 (dt, J = 8.1, 0.8 Hz,
1
7
2
1
H), 7.18-7.09 (m, 4H), 7.05 (dd, J = 8.2, 7.0 Hz, 1H), 7.01-6.95 (m, 3H), 4.56 (t, J =
.2 Hz, 2H), 3.29-3.26 (m, 4H) 3.01 (t, J = 7.1 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.71-
.61 (m, 6H). 13C NMR (126 MHz, DMSO-d
) δ 155.0, 151.4, 150.4, 141.1, 137.3,
6
36.3, 128.7 (2C), 128.2 (2C), 127.4, 126.7, 122.6, 121.0, 118.4, 118.3, 118.1, 114.3,
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12.6, 111.5, 86.2, 59.0, 52.3 (2C), 50.3, 50.0 (2C), 37.2, 22.4. LC-MS: (m/z) calc.:
+
475.3; found: 476.2 [M+H] . Purity: 97%.
4-(4-(2-(1H-Indole-3-yl)ethyl)piperazine-1-yl)-5-(3-phenylpropyl)-5H-pyrrolo[3,2-
0
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d]pyrimidine-7-carbonitrile (55) Pale yellow crystals; yield: 16%. H NMR (500 MHz,
DMSO-d
6
) δ 10.75 (s, 1H), 8.62 (s, 1H), 8.54 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.33 (d,
J = 8.1 Hz, 1H), 7.23 (t, J = 7.5 Hz, 2H), 7.16 (d, J = 2.1 Hz, 1H), 7.14 (t, J = 7.4 Hz,
1
H), 7.09 (d, J = 7.0 Hz, 2H), 7.05 (m, 1H), 6.97 (m, 1H), 4.33 (t, J = 7.3 Hz, 2H) 3.27-
.23 (m, 6H), 2.87 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.59-2.51 (m, 4H), 2.09
3
1
3
(
p, J = 7.5 Hz, 2H). C NMR (126 MHz, DMSO-d
6
) δ 155.0, 151.4, 150.4, 140.9, 140.5,
136.3, 128.5 (2C), 128.3 (2C), 127.3, 126.2, 122.6, 120.9, 118.4, 118.3, 118.0, 114.3,
112.6, 111.5, 86.2, 58.8, 52.2 (2C), 50.1 (2C), 48.6, 32.0, 32.0, 22.5. LC-MS: (m/z)
+
calc.: 489.3; found: 490.3 [M+H] . Purity: 95%.
4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-yl)-6,7,8,9-tetrahydropyrimido[4,5-
1
b]indolizine-10-carbonitrile (56) White crystals; yield: 46%. H NMR (500 MHz, DMSO-
d
6
) δ 8.45 (s, 1H), 6.88 (d, J = 1.5 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 6.77 (dd, J = 7.9,
1.5 Hz, 1H), 5.98 (s, 2H), 4.33 (t, J = 5.6 Hz, 2H), 3.45 (s, 2H), 3.36-3.29 (m, 4H), 3.10
1
3
(t, J = 6.4 Hz, 2H), 2.61-2.50 (m, 4H), 1.98-1.84 (m, 4H). C NMR (126 MHz, DMSO-
d
6
) δ 154.3, 151.2, 150.9, 150.2, 147.4, 146.4, 131.9, 122.2, 118.2, 114.4, 109.3,
108.0, 100.9, 83.6, 61.9, 52.1 (2C), 50.4 (2C), 46.1, 22.9, 22.6, 18.4. LC-MS: (m/z)
+
calc.: 416.20; found: 490.3 [M+H] . Purity: 98%.
4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-yl)-7,8,9,10-tetrahydro-6H-
pyrimido[4',5':4,5]pyrrolo[1,2-a]azepine-11-carbonitrile (57) Pale yellow crystals; yield:
1
35%. H NMR (500 MHz, DMSO-d
6
) δ 8.46 (s, 1H), 6.88 (d, J = 1.5 Hz, 1H), 6.84 (d, J
=
2
7.8 Hz, 1H), 6.77 (dd, J = 7.9, 1.6 Hz, 1H), 5.98 (s, 2H), 4.54-4.40 (m, 2H), 3.45 (s,
H), 3.35-3.29 (m, 4H), 3.12-3.04 (m, 2H), 2.61-2.49 (m, 4H), 1.89-1.80 (m, 2H), 180-
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68 m, 2H). C NMR (126 MHz, DMSO-d ) δ 156.6, 153.7, 151.1, 149.6, 147.4, 146.3,
6
4
5
6
7
8
9
1
1
1
1
1
1
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1
1
2
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2
2
2
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4
4
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5
5
5
5
5
5
5
5
5
6
1
31.9, 122.2, 117.9, 114.6, 109.3, 108.0, 100.9, 85.3, 61.8, 51.9 (2C), 49.8 (2C), 47.4,
+
29.9, 27.9, 27.2, 25.6. LC-MS: (m/z) calc.: 430.21; found: 431.2 [M+H] . Purity: 100%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
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4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-yl)-5-phenyl-5H-pyrrolo[3,2-
1
d]pyrimidine-7-carbonitrile (58) Yellow crystals; yield: 37%. H NMR (500 MHz, DMSO-
d
6
, δ 8.80 (s, 1H), 8.64 (s, 1H), 7.58-7.46 (m, 5H), 707 (s, 1H), 6.97 (s, 1H), 6.90 (s,
13
1H), 6.06 (s, 1H), 4.17-3.97 (m, 2H), 3.73-3.40 (m, 4H), 315-2.96 (m, 4H)). C NMR
(
(
126 MHz, DMSO-d , δ 151.7, 151.4, 147.5, 141.7, 137.5, 129.5 (3C), 128.6, 125.4
6
3C), 115.5, 113.9, 101.6, 88.2, 49.1 (2C), 45.2 (2C)). LC-MS: (m/z) calc.: 438.2; found:
+
439.1 [M+H] . Purity: 98%.
4-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-yl)-5-phenethyl-5H-pyrrolo[3,2-
1
d]pyrimidine-7-carbonitrile (59) White crystals; yield: 20%. H NMR (500 MHz, DMSO-
d
6
) δ 8.55 (s, 1H), 8.48 (s, 1H), 7.17-7.11 (m, 3H), 6.98-6.95 (m, 2H), 6.87 (d, J = 1.4
Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 6.76 (dd, J = 7.9, 1.5 Hz, 1H), 5.98 (s, 2H) ,4.52 (t, J
=
4
7.2 Hz, 2H), 3.44 (s, 2H), 3.26-3.20 (m, 4H), 3.00 (t, J = 7.1 Hz, 2H), 2.57-2.51 (m,
1
3
H). C NMR (126 MHz, DMSO-d ) δ 154.9, 151.4, 150.5, 147.4, 146.4, 141.1, 137.3,
6
131.8, 128.7 (2C), 128.3 (2C), 126.8, 122.3, 118.1, 114.3, 109.3, 108.3, 101.0, 86.2,
1.8, 52.0 (2C), 50.3, 50.0 (2C), 37.2. LC-MS: (m/z) calc.: 466.2; found: 467.3 [M+H]+.
6
Purity: 99%.
4-(4-(Furan-2-carbonyl)piperazine-1-yl)-6,7,8,9-tetrahydropyrimido[4,5-b]indolizine-
1
10-carbonitrile (60) Yellow crystals; yield: 37%. H NMR (500 MHz, DMSO-d
6
) δ 8.49
(
s, 1H), 7.85 (dd, J = 1.7, 0.7 Hz, 1H), 7.03 (dd, J = 3.5, 0.7 Hz, 1H), 6.64 (dd, J = 3.4,
1.8 Hz, 1H), 4.40 (t, J = 5.6 Hz, 2H), 3.94-3.82 (m, 4H), 3.39 (t, J = 5.0 Hz, 4H), 3.13
13
(
t, J = 6.4 Hz, 2H), 2.02-1.86 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 158.7, 153.9,
6
151.2, 151.1, 150.3, 147.0, 144.9, 118.2, 115.9, 114.4, 111.5, 83.6, 60.4, 46.0 (2C),
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