Pyrrolopyrimidine Derivatives as Novel Inhibitors of Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1)

Article abstract of DOI:10.1021/acs.jmedchem.5b01644

Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC₅₀ values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).

Full text of DOI:10.1021/acs.jmedchem.5b01644

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Article
Pyrrolopyrimidine-derivatives as novel inhibitors of
Multidrug Resistance- associated Protein 1 (MRP1, ABCC1)
Sven Marcel Schmitt, Katja Stefan, and Michael Wiese
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01644 • Publication Date (Web): 04 Mar 2016
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Pyrrolopyrimidine derivatives as novel inhibitors of Multidrug Resistance-
associated Protein 1 (MRP1, ABCC1)
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Sven Marcel Schmitt , Katja Stefan , Michael Wiese*
University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn,
Germany
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Eꢀmail: mwiese@uniꢀbonn.de, phone: +49 228 73 5212
†
These authors contributed equally.
Key words:
ABC transporter; MRP1; ABCC1; MDR; pyrrolopyrimidine; inhibitor
Abstract: Five series of pyrrolo[3,2ꢀd]pyrimidines were synthesized and evaluated
with respect to potency and selectivity toward Multidrug Resistanceꢀassociated
Protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome
multidrug resistance in cancer patients. We investigated differently substituted
pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small
cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation
assay. New compounds with high potency and selectivity were identified.
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Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be
beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased
activity. Aliphatic and aliphaticꢀaromatic variations at position 5 and 6 revealed
compounds with IC50 values in high nanomolar range. All investigated compounds
had low affinity toward Pꢀglycoprotein (Pꢀgp, ABCB1). Pyrrolopyrimidines with small
substituents showed moderate inhibition against Breast Cancer Resistance Protein
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Introduction. The 190 kDa Multidrug Resistanceꢀassociated Protein 1 (MRP1,
ABCC1) is a member of the ATPꢀbinding cassette (ABC) superfamily of transport
proteins. Besides Pꢀgp and BCRP it represents an important transporter in cancer
research, conferring resistance to common chemotherapeutic agents. This
phenomenon is called multidrug resistance (MDR) as many structurally unrelated
drugs are affected like the Vinca alkaloids vincristin and vinblastin, the anthracyclines
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doxorubicin, daunorubicin or epirubicin as well as the intercalating agent
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mitoxantrone or the podophyllotoxine etoposid.
Utilizing the energy of ATP
hydrolysis, this active transport leads to decreased intracellular drug accumulation
2,3,4,7,8
and often involves coꢀtransportation of glutathione (GSH).
MRP1 is reported to
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be nearly ubiquitously expressed in human tissue
as well as in different cancer
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types like small (SCLC) and nonꢀsmall (NSCLC) cell lung cancer
, colon
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carcinomas or leukemia.
It is associated with reduced retention of xenobiotics
and chemotherapeutics since these are extruded as glucuronate or sulfate
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conjugates.
The pharmacological function of MRP1 might be the regulation of
cellular distress as it has high affinity toward endogenous mediators connected to
oxidative stress and cell apoptosis like leukotriene C (LTC ), GSH and its oxidized
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4
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form GSSG.
Many structurally diverse compounds have been identified to inhibit MRP1 since its
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discovery and depiction between 1987 and 1992 by Cole et al.
The coꢀ
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transportation of the ATPase modulator GSH
with many cytostatic compounds
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led to glutathionꢀconjugate analogs as inhibitors of MRP1. Related to LTC , another
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endogenous substrate of MRP1, the leukotriene D
4 4
(LTD ) receptor antagonist MKꢀ
571 (Verlukast, Figure 1) was found to reverse vincristine resistance at a 30 ꢁM
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concentration in the H69 AR cell line overexpressing MRP1. Similar results were
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obtained by ONOꢀ1078 (Pranlukast), another leukotriene receptor antagonist.
Reversal of MDR affecting doxorubicin and paclitaxel was also observed with the
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pyridine analog PAKꢀ140P in different sarcoma and leukemia cell lines.
Especially drugs from clinical pharmacology were evaluated as MRP1 inhibitors, for
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example the anion transport inhibitor probenecid, the nonsteroidal antiꢀinflammatory
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drug indometacin (Figure 1)
or the immunosuppressive agent cyclosporine A and
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its chemosensitizing derivative PSCꢀ833 (Valspodar).
The MRP1ꢀmediated
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transport of cytostatics is also affected by the calcium antagonist verapamil
and
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different reverse transcriptase inhibitors.
Due to the structural and functional
analogy of MRP1 and Pꢀgp, many compounds were discovered due to their ability to
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inhibit the latter transport protein. Modifications of their scaffolds resulted in new
inhibitors designed to affect MRP1 selectively and to elucidate the structureꢀactivity
relationships of different partial structures. Rosenbaum et al. reported on
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indometacinꢀanalogs to increase cell death in combination with doxorubicin.
Another approach dealt with the development of verapamil analogs that affect LTC
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and GSH transport, after verapamil was found to stimulate GSH transport.
Other compounds were developed as dual inhibitors of Pꢀgp and MRP1, e.g. the
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quinolone derivative MS209, chalcogenpyrylium compounds or quinazolinones
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(Figure 1). The latter were derived from a tricyclic isoxazole scaffold
by Elli
Lilly, who reported already on the drug export inhibiting cromen derivative LY294002
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in 1996. The pipecolinate derivative Biricodar (VXꢀ710) was evaluated in clinical
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trials regarding its dual inhibition of Pꢀgp and MRP1.
Natural products were also
found to be inhibitors of MRP1, e.g. Agosterol A, a compound isolated from the
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marine sponge Spongia sp.
as well as flavonoids, which were firstly known as Pꢀ
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gp inhibitors.
Further investigations of flavonoids like apigenin proved these
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compounds to be potent MRP1 ATPase stimulators.
Synthesis of flavonoidꢀ
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analogs provided potent inhibitors of this transport protein.
α,βꢀunsaturated
carbonyl compounds like curcumin and derivatives were also found to have inhibitory
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activity against MRP1. Leyers and Häcker et al. showed in 2008 and 2009,
respectively, that compounds bearing a thiourea moiety affect transport of calcein AM
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in the 2008/MRP1 cell line.
More recently it was reported that the tyrosine kinase
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inhibitors (TKI) ibrutinib and lapatinib also affect MRP1 in an inhibitory way.
TKIs
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have only been known as inhibitors of BCRP before.
Wang et al., which had reported on quinazolinones to be potent inhibitors in
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nanomolar range, found pyrroloꢀ and indolopyrimidines (Figure 1) as raloxifeneꢀ
derived compounds to be the most active inhibitors toward MRP1 known until
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now.
The aim of our investigation was to identify important substructures of the
pyrrolopyrimidine scaffold and to analyze the effect of different substitutions at
positions 4, 5 and 6 regarding length and size of side chains.
Results and Discussion
Chemistry
All compounds were prepared as illustrated in schemes 1 and 2. The malononitrile
derivatives were derived by nucleophilic substitution of malononitrile with Oꢀ
methylated lactams (2a-b) as well as nucleophilic substitution of ethoxymethyleneꢀ
malononitrile with differently substituted primary amines (7a-i). Cyclization with ethyl
bromoacetate yielded pyrrol derivatives 3a-b and 8a-i. Addition of dimethyl
formamide dimethyl acetale provided compounds 4a-b and 9a-i. Cyclization was
conducted using gaseous ammonia in ethanol to build up the pyrimidine ring system
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of compounds 5a-b and 10a-i. Aromatization was accomplished by treating the
lactam structureꢀbearing substances with phosphoryl chloride giving the chlorinated
precursors 6a-b and 11a-i. The desired compounds 12-38 were obtained by
nucleophilic substitution at position 4 by 1Hꢀpiperazineꢀ and different primary amines
using microwaveꢀassisted synthesis. The identity of intermediates 2a-b, 3a-b, 4a-b,
5a-b, 6a-b as well as compounds 7a-i, 8a-i, 9a-i, 10a-i and 11a-i was confirmed
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using H NMR spectroscopy. The purity of the desired compounds 12-38 was
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determined using H and C NMR spectroscopy as well as LCꢀMS analysis.
Biological Investigation
Calcein AM and Daunorubicin Accumulation Assays. In order to analyze the influence
of different substituents of the pyrrolopyrimidine structure we investigated variably
substituted derivatives belonging to various structural classes.
Our first aim was to analyze the contribution of the aliphatic linker between the
piperazine substructure at position 4 and the terminal phenyl ring with regard to
MRP1 inhibition. Compound 12 (Figure 1) served as starting structure bearing a
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phenethyl group. This compound has already been described by Wang et al. and
possessed an IC ꢀvalue of 0.370 ꢁM in the calcein AM assay and 0.197 ꢁM in the
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daunorubicin assay, respectively. This makes it a rather potent inhibitor of MRP1
compared to standard inhibitors like indomethacin or cyclosporine A (Figure S1 and
S2).
A stepwise reduction of the phenethyl residue to a benzyl (13) and phenyl (15)
substituent lowered the activity for each reduction about threeꢀfold, as shown in
Figure 2 and table 1. Thus the ethylene linker is well accepted in contrast to the
shortened linkers. The correlation of the predicted partition coefficient (log P) and the
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activity of the compounds (Figure 3) shows that high lipophilicity is beneficial for
MRP1 inhibition. Compound 14 bearing a diphenylmethylpiperazine side chain
represents a downward outlier. It shows a potency twoꢀfold less than the
benzylpiperazine derivative 13 although it has a high calculated log P value.
Obviously, the sterically demanding diphenylmethyl residue is tolerated but
contributes less to activity than could be expected. But the necessity of a lipophilic
terminal part of the substituent becomes obvious from the comparison of compounds
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16 and 17, having a methylpiperazine and piperazine substructure, respectively.
Therefore a lipophilic group containing an aromatic ring system seems to be required
for an effective MRP1 inhibition. Figure 4 shows a comparison of representative
doseꢀresponse curves of compounds 12, 16 and 17.
The second compound set consisted of four substances (18-21) in which compound
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1
9 has been described by Wang et al. (Figure 1) before . The piperazine linker was
replaced by an amino group, resulting in decreased activity values. The correlation of
inhibitory activity and lipophilicity is obvious, showing that the downsizing of the
aliphatic linker leads to less lipophilic properties and simultaneously to a decrease of
MRP1 inhibition of about one order of magnitude (table 1).
The next step was to see if similar changes in the side chains at positions 5 and 6
have similar results regarding MRP1 activity. The enlargement of the cyclohexyl to a
cycloheptyl ring of compounds 22ꢀ25 (third compound set, table 2) showed that all
cycloheptyl derivatives are inferior compared to their cyclohexyl counterparts (12ꢀ15).
Nevertheless, compound 22 has still high affinity toward MRP1 with an IC50 value of
0.833 ꢁM in the calcein AM assay and 0.643 ꢁM in the daunorubicin assay. The
replacement of the cyclic ring system by aliphatic side chains makes up the fourth
compound set (26ꢀ30). These pyrrolopyrimidine derivatives proved to be potent
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MRP1 inhibitors with IC50 values in high nanomolar range as it is summarized in table
2. Compound 30 was equally potent as standard compound 12. With respect to the
activityꢀlog P relationship, all compounds form a cluster without correlation.
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The fifth compound set was destined to determine the influence of large residues at
position 5. Therefore, the aliphatic residue was enlarged with a benzene ring at the
end of the chain. Within this set of compounds no. 31 bearing a phenyl residue was
the most active one possessing an IC50 value of 0.750 ꢁM in the calcein AM assay
and 0.340 ꢁM in the daunorubicin assay. All compounds are potent inhibitors of
MRP1 in the low micromolar or high nanomolar range (table 3) analogous to
compound set 4. No correlation was observed with regard to lipophilicity and calcein
AM or daunorubicin activity data.
With regard to the molecular weight of all compounds, one can observe a correlation
relating to MRP1 activity as can be seen in Figure 5. Concerning pyrrolopyrimidines,
hydrophobic residues influencing the partition coefficient toward high lipophilicity are
preferred up to 400 Da. The increase of molecular weight over this point did not lead
to more active compounds, but kept the inhibitory level around 1 ꢁM.
The results of both accumulation assays are summarized in Figure 6, showing a
linear correlation of activities toward MRP1 within two and half orders of magnitude.
Calcein AM Accumulation Assay to Screen for Pꢀgp Inhibition. Because of the
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functional and structural analogy between MRP1 and Pꢀgp we screened all
compounds for their capability to inhibit the latter transport protein. The results are
depicted in Figure 7 and tables 1 to 3. All compounds were poor inhibitors of Pꢀgp
with IC50ꢀvalues higher than the first generation inhibitor verapamil and about
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hundred fold less potent than tariquidar or elacridar in the same assay.
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Pheophorbide A Accumulation Assay to Determine the Inhibitory Activity toward
BCRP. All compounds were screened for their capability to inhibit BCRP to ensure
selectivity toward MRP1. As can be seen in Figure 8, most compounds from sets one
to three show inhibition of BCRP of more than 25% at 10 µM in comparison to 10 ꢁM
Ko143 and were further characterized by generating full doseꢀresponse curves. The
calculated IC50ꢀvalues are given in tables 1 and 2. Activity toward BCRP was not
influenced by shortening of the side chain length in set one (12-15). Only compound
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14 showed less potency as it contains a bulky diphenylmethylpiperazine residue.
Analogous results were partially obtained for the cycloheptyl counterparts (22ꢀ25),
although compound 25 was inactive in contrast to compound 15. The
methylpiperazine residue bearing compound 16 shows even better inhibitory activity
with an IC50 value of 4.25 ꢁM and represents a good scaffold for BCRP inhibition
(table 1). Compound 17 is a downward outlier though its structural similarity to
compound 16. The replacement of the piperazine by an amino group in compound
set two (18ꢀ21) did not change the activity except of the inactive phenethylamino
derivative 19. The substances of the third, fourth and fifth compound set revealed no
BCRP activity.
MTT Assay to Determine Cytotoxicity of Selected Compounds. To evaluate the
cytotoxic potential of the pyrrolopyrimidines we investigated representatives of all
compound sets with regard to toxicity on H69 AR cells. As Figure 9 shows, nearly all
representatives show only a slight influence on cell viability at 10 ꢁM. The only
exception is compound 31 from the fifth compound set that reduced the cell viability
by 40%. The GI50 values of selected test compounds are depicted in table 4.
MTTꢀefficacy Assay to Determine Capability of Compounds to Restore Daunorubicin
Sensitivity. Due to their mostly nontoxic characteristics, potent and less potent
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representatives of all compound sets were investigated regarding their capability to
reverse multidrug resistance mediated by MRP1. For this purpose the daunorubicin
MTTꢀefficacy assay was conducted. All of the chosen compounds were able to
reverse multidrug resistance, as can be seen in table 5. Compound 31 was most
efficient in reversing MDR, but its intrinsic toxicity (Figure 9) might have contributed to
this effect. Compound 30 proved to be nonꢀtoxic and increased the cytotoxicity of
daunorubicin in H69 AR cells tenꢀfold, as depicted in Figure 10.
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Conclusion
We elucidated the structureꢀactivity relationship of pyrrolopyrimidines with variations
at positions 4, 5 and 6 regarding the three major transport proteins involved in
multidrug resistance of cancer cells. Long phenylalkyl residues linked to piperazine at
position 4 are preferred as compounds with shortened side chains show reduced
activity. The enlargement of the aliphatic side chain at position 5 increased the
capability to inhibit MRP1 with great potency, as compound 30 demonstrates. Even
large aromatic residues are accepted at this position. Especially small molecules of
the pyrrolopyrimidine compound sets like compounds 15, 16 or 18 are good
representatives of dual inhibitors for MRP1 and BCRP or future BCRP inhibitors.
Except of compound set 5 (31ꢀ38) all compound proved to be nonꢀtoxic, showing that
the decrease in daunorubicin resistance in H69 AR cells correlates with their MRP1
inhibition capability.
Experimental Section
Chemistry. Materials. All chemicals were supplied by Acros Organics (Geel,
Belgium), Alfa Aesar (Karlsruhe, Germany), Applichem GmbH (Darmstadt,
Germany), Fisher Scientific GmbH (Waltham, MA, USA), Merck Millipore (Billerica,
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MA, USA), SigmaꢀAldrich (St. Louis, MO, USA) and VWR International GmbH
(Darmstadt, Germany) and used for synthesis without further purification. Analytical
thin layer chromatography with silica gel F254 coated aluminum plates (Merck
Millipore) were used to monitor reaction progress using methylene chloride/acetone
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
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
18:1 and 9:1, respectively) or methylene chloride/acetone/methanol (9:1:1) as eluent
and an UV cabinet for compound detection at 254 nm. All compounds were purified
with column chromatography using silica gel 60 (43ꢀ60 ꢁm, Merck Millipore) with
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 9:1:1, in
1
13
each case 200 mL). Identity of intermediates was determined by H and C NMR
1
spectroscopy. The purity of the desired compounds 12-38 was confirmed by H and
1
3
C NMR spectroscopy as well as LCꢀMS analysis. All NMR spectra were recorded
in DMSOꢀd on a Bruker Advance 500 MHz (500/126 MHz) and chemical shifts (δ)
are expressed as ppm calibrated to the solvent signal ( H NMR δ 2.50; C NMR δ
6
1
13
13
3
9.5).
C signals were assigned employing distortion less enhancement by
polarization transfer (DEPT) and attached proton test (APT) techniques and spin
multiplicities are depicted as singlet (s), doublet (d), doublet of doublets (dd), doublet
of triplets (dt), triplet of doublets (td), triplet (t), quartet (q), quintet (quint) and multiplet
(m). LCꢀMS analysis was performed using an Agilent 1100 series with photo diode
array (DAD) detector (Agilent Technologies, Santa Clara, CA, USA) and a Nucleodur
column 100ꢀ5 C18 (MachereyꢀNagel, 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.
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General Procedure for the Preparation of Compounds 6aꢀb. The title compounds
7
7
were prepared as described in the literature with minor modifications as depicted in
schemes 1 and 2. A solution of piperidinꢀ2ꢀone or azepanꢀ2ꢀone in benzene was
heated and an equimolar amount of dimethyl sulfate was added dropwise. The
reaction mixture was refluxed for 3 to 6 hours. To eliminate excess dimethyl sulfate, a
4 M solution of sodium hydroxide was added after cooling to room temperature and
stirred for 30 minutes. The title compounds were extracted three times with 50 mL
benzene from the aqueous phase and dried over magnesium sulfate. After filtration
and solvent evaporation, to the resultant oil containing 6ꢀmethoxyꢀ2,3,4,5ꢀ
tetrahydropyridine (1a) or 7ꢀmethoxyꢀ3,4,5,6ꢀtetrahydroꢀ2Hꢀazepine (1b) a saturated,
equimolar solution of malononitrile in ethanol was added under stirring. After 30
minutes, the precipitate was rinsed with ethanol and diethyl ether and dried. The
0
1
2
3
4
5
6
7
8
9
0
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9
0
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8
9
0
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7
8
9
0
resultant
2ꢀ(piperidinꢀ2ꢀylidene)malononitrile
(2a)
or
2ꢀ(azepanꢀ2ꢀ
ylidene)malononitrile (2b) was dissolved in dimethyl formamide and potassium
carbonate was added. The reaction mixture was heated to 100 °C and an equimolar
amount of ethyl bromoacetate was added dropwise. After a reaction time of 3 to 6
hours, the mixture was given to ice cold water (200 mL) under stirring and filtered
after 1 hour. The resultant ethyl 2ꢀaminoꢀ1ꢀcyanoꢀ5,6,7,8ꢀtetrahydroindolizineꢀ3ꢀ
carboxylate (3a) or ethyl 2ꢀaminoꢀ1ꢀcyanoꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀpyrrolo[1,2ꢀ
a]azepineꢀ3ꢀcarboxylate (3b) was used without further purification. Three to five
equivalents of dimethyl formamide dimethyl acetale were added to a solution of
compounds (3a) or (3b) in dimethyl formamide and heated to 100 °C. After a reaction
time of 2 to 5 hours, the solvent was evaporated and the resultant viscous liquid was
stirred under petroleum ether until full crystallization, providing ethyl (E)ꢀ1ꢀcyanoꢀ2ꢀ
(((dimethylamino)methylene)amino)ꢀ5,6,7,8ꢀtetrahydroindolizineꢀ3ꢀcarboxylate
(4a)
or ethyl (E)ꢀ1ꢀcyanoꢀ2ꢀ(((dimethylamino)methylene)amino)ꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀ
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pyrrolo[1,2ꢀa]azepineꢀ3ꢀcarboxylate (4b), which were washed with petroleum ether
and dried. The compounds (4a) or (4b) were solved in ethanol, heated to reflux and
exposed to gaseous ammonia for 5 hours. The solvent was evaporated and a
solution of 10% sodium hydroxide was added under stirring. After 30 minutes, the
alkaline solution was neutralized with acetic acid. The resultant precipitate of 4ꢀoxoꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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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
3,4,6,7,8,9ꢀhexahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
,6,7,8,9,10ꢀhexahydroꢀ3Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀa]azepineꢀ11ꢀcarbonitrile
5b) was filtered off and rinsed with water. The dried compounds (5a) or (5b) were
(5a)
or
4ꢀoxoꢀ
4
(
added to 20 mL of phosphoryl chloride and heated to reflux overnight. After cooling to
room temperature, the reaction mixture was given to ice cold water under stirring.
The resultant precipitate of compounds (6a) or (6b) was filtered off and washed with
water before drying.
1
2
ꢀ(piperidinꢀ2ꢀylidene)malononitrile (2a). White needles; yield 77%. H NMR (500
6
MHz, DMSOꢀd ) δ 9.01 (s, 1H), 3.23 (t, J = 6.0 Hz, 2H), 2.55 (t, J = 6.0 Hz, 2H), 1.74ꢀ
13
159 (m, 4H). C NMR (126 MHz, DMSOꢀd
6
) δ 171.5, 117.2, 116.0, 45.3, 42.3, 26.8,
20.7, 18.0.
1
2
ꢀ(azepanꢀ2ꢀylidene)malononitrile (2b). White needles; yield 55%. H NMR (500
MHz, DMSOꢀd ) δ 8.93 (s, 1H), 3.41ꢀ3.38 (m, 2H), 2.67ꢀ2.64 (m, 2H), 1.70ꢀ1.64 (m,
6
13
2
6
H), 1.58ꢀ1.55 (m, 2H), 1.52ꢀ1.46 (m, 2H). C NMR (126 MHz, DMSOꢀd ) δ 177.1,
117.4, 115.9, 46.9, 44.8, 30.9, 29.2, 27.8, 24.2.
Ethylꢀ2ꢀaminoꢀ1ꢀcyanoꢀ5,6,7,8ꢀtetrahydroindolizineꢀ3ꢀcarboxylate (3a). White powder;
1
quantitative yield. H NMR (500 MHz, DMSOꢀd ) δ 5.75 (s, 2H), 4.19 (q, J = 7.1 Hz,
6
2
2
1
H), 4.05 (t, J = 6.1 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 1.88ꢀ1.83 (m, 2H), 1.74ꢀ1.69 (m,
13
6
H), 1.25 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 160.7, 146.0, 142.5,
15.2, 104.0, 79.3, 59.0, 45.7, 22.4, 22.3, 18.3, 14.6.
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Ethylꢀ2ꢀaminoꢀ1ꢀcyanoꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀpyrrolo[1,2ꢀa]azepineꢀ3ꢀcarboxylate
1
(
3b). Pale yellow powder; yield 55%. H NMR (500 MHz, DMSOꢀd ) δ 5.67 (s, 2H),
6
4
1
1
.49ꢀ4.40 (m, 2H), 4.21 (q, J = 7.1 Hz, 2H), 2.77ꢀ2.73 (m, 2H), 1.76ꢀ1.70 (m, 2H),
13
6
.60ꢀ1.54 (m, 4H), 1.25 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSOꢀd ) δ 160.7,
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
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1
2
3
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6
7
8
9
0
48.6, 145.3, 115.3, 104.1, 80.7, 59.2, 46.1, 29.8, 27.5, 26.1, 25.6, 14.6.
Ethylꢀ(E)ꢀ1ꢀcyanoꢀ2ꢀ(((dimethylamino)methylene)amino)ꢀ5,6,7,8ꢀtetrahydroindolizineꢀ
1
3ꢀcarboxylate (4a). Yellow needles; yield: 75%. H NMR (500 MHz, DMSOꢀd
6
) δ 7.52
(s, 1H), 4.12 (t, J = 6.1 Hz, 2H), 4.07 (q, J = 7.1 Hz, 2H), 2.97 (s, 3H), 2.91 (s, 3H),
2.75 (t, J = 6.4 Hz, 2H), 1.91ꢀ1.85 (m, 2H), 1.77ꢀ1.71 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).
13
6
C NMR (126 MHz, DMSOꢀd ) δ 160.5, 156.2 (2C), 148.7, 141.6, 115.7, 110.3, 86.5,
59.2, 46.0, 33.8, 22.5, 22.5, 18.4, 14.0.
Ethylꢀ(E)ꢀ1ꢀcyanoꢀ2ꢀ(((dimethylamino)methylene)amino)ꢀ6,7,8,9ꢀtetrahydroꢀ5Hꢀ
pyrrolo[1,2ꢀa]azepineꢀ3ꢀcarboxylate (4b). Yellow powder; yield: 40%. Used without
further purification and characterization.
4ꢀoxoꢀ3,4,6,7,8,9ꢀhexahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
(5a).
White
1
powder; yield: 68%. H NMR (500 MHz, DMSOꢀd
6
) δ 12.21 (s, 1H), 7.90 (s, 1H), 4.35
1
3
(t, J = 6.4 Hz, 2H), 2.95 (t, J = 6.4 Hz, 2H), 1.99ꢀ1.93 (m, 2H), 1.87ꢀ1.80 (m, 2H).
C
NMR (126 MHz, DMSOꢀd ) δ 153.2, 146.3, 145.5, 144.8, 116.7, 114.3, 84.0, 45.6,
6
22.4, 21.8, 18.3.
4ꢀoxoꢀ4,6,7,8,9,10ꢀhexahydroꢀ3Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀa]azepineꢀ11ꢀ
1
carbonitrile (5b). White powder; yield 12%. H NMR (500 MHz, DMSOꢀd
6
) δ 12.30 (s,
1
3
1H), 4.82ꢀ4.69 (m, 2H), 2.01ꢀ2.95 (m, 2H), 1.85ꢀ1.79 (m, 2H), 1.72ꢀ1.63 (m, 4H).
C
NMR (126 MHz, DMSOꢀd
6
) δ 153.5, 152.2, 144.8, 144.5, 117.0, 114.5, 85.5, 47.1,
30.0, 27.8, 26.4, 25.7.
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4ꢀchloroꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
(6a).
White
1
needles; yield 42%. H NMR (500 MHz, DMSOꢀd ) δ 8.71 (s, 1H), 4.52 (t, J = 6.2 Hz,
6
13
2H), 3.15 (t, J = 6.4 Hz, 2H), 2.09ꢀ2.03 (m, 2H), 1.91ꢀ1.85 (m, 2H). C NMR (126
6
MHz, DMSOꢀd ) δ 154.8, 151.3, 150.8, 142.1, 123.5, 113.4, 83.8, 46.0, 23.6, 21.8,
10
11
12
13
14
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43
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47
48
49
50
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53
54
55
56
57
58
59
60
1
7.8.
4ꢀchloroꢀ7,8,9,10ꢀtetrahydroꢀ6Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀa]azepineꢀ11ꢀ
1
carbonitrile (6b). Brown powder; yield 71%. H NMR (500 MHz, DMSOꢀd ) δ 8.73 (s,
6
13
1H), 4.80ꢀ4.75 (m, 2H), 3.20ꢀ3.16 (m, 2H), 1.90ꢀ1.71 (m, 6H). C NMR (126 MHz,
DMSOꢀd
6
) δ 160.1, 150.8, 150.8, 141.5, 123.6, 113.6, 85.4, 47.00, 29.3, 27.1, 26.9,
25.2.
General procedure for the preparation of compounds 11a-i. 1.1 equivalents of the
amine were added to a saturated solution of 2ꢀ(ethoxymethylene)malononitrile in
ethanol under stirring. After 5ꢀ60 minutes the resulting precipitate was rinsed with
ethanol and diethyl ether yielding compounds (7a) or (7b). After filtration and drying,
the compounds (7a) or (7b) were solved in dimethyl formamide and heated to 100
°C. An equimolar amount of ethyl bromoacetate was added dropwise, followed by
reactions and procedures as described above, resulting in the title compounds.
1
2ꢀ((methylamino)methylene)malononitrile (7a). Orange needles; yield: 63%. H NMR
6
(500 MHz, DMSOꢀd ) δ 8.91 (s, 1H), 7.86 (s, 1H), 2.96 (s, 3H).
1
2ꢀ((ethylamino)methylene)malononitrile (7b). Yellow needles; yield: 54%. H NMR
(500 MHz, DMSOꢀd
6
) δ 9.12 (s, 1H), 7.89 (d, J = 14.8 Hz, 1H), 3.28ꢀ3.24 (m, 2H),
1.11 (t, J = 7.2 Hz, 3H).
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2ꢀ((propylamino)methylene)malononitrile (7c). Yellow needles; quantitative yield. H
NMR (500 MHz, DMSOꢀd ) δ 9.10 (s, 1H), 7.89 (d, J = 14.3 Hz, 1H), 3.21 (q, J = 7.2
6
Hz, 2H), 1.50 (dt, J = 14.6, 7.3 Hz, 2H), 0.82 (t, J = 7.4 Hz, 3H).
0
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5
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7
8
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1
2
ꢀ((isopropylamino)methylene)malononitrile (7d). Orange needles; yield: 88%. H
6
NMR (500 MHz, DMSOꢀd ) δ 9.12 (s, 1H), 7.88 (d, J = 14.6 Hz, 1H), 3.67ꢀ3.57 (m,
1H), 1.16 (d, J = 6.6 Hz, 6H).
2ꢀ((cyclopropylamino)methylene)malononitrile (7e). Yellow needles; yield: 60%. Used
without further purification and characterization.
1
2
ꢀ((phenylamino)methylene)malononitrile (7f) Yellow needles; yield: 78%. H NMR
(
500 MHz, DMSOꢀd
6
) δ 11.09 (s, 1H), 8.49 (s, 1H), 7.42 (d, J = 7.7 Hz, 2H), 7.37 (t, J
=
7.4 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H).
1
2
ꢀ((benzylamino)methylene)malononitrile (7g). Yellow needles; yield: 61%. H NMR
(
500 MHz, DMSOꢀd ) δ 9.58 (s, 1H), 8.08 (s, 1H), 7.40ꢀ7.35 (m, 2H), 7.34ꢀ7.28 (m,
6
3H), 4.44 (s, 2H).
1
2ꢀ((phenethylamino)methylene)malononitrile (7h). White needles; yield: 61%.
H
NMR (500 MHz, DMSOꢀd
6
) δ 9.16 (s, 1H), 7.76 (s, 1H), 7.33ꢀ7.29 (m, 2H), 7.24ꢀ7.21
(m, 1H), 7.21ꢀ7.18 (m, 2H), 3.49 (t, J = 7.1 Hz, 2H), 2.82 (t, J = 7.3 Hz, 2H).
2
ꢀ(((3ꢀphenylpropyl)amino)methylene)malononitrile (7i). Yellow powder; yield: 70%.
1
6
H NMR (500 MHz, DMSOꢀd ) δ 9.10 (s, 1H), 7.88 (s, 1H), 7.30ꢀ7.25 (m, 2H), 7.21ꢀ
7.16 (m, 3H), 3.26 (t, J = 7.1 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H), 1.86ꢀ1.79 (m, 2H).
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8a). Brown powder; yield:
1
5
3%. H NMR (500 MHz, DMSOꢀd ) δ 7.50 (s, 1H), 5.71 (s, 2H), 4.21 (q, J = 7.1 Hz,
6
2H), 3.69 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).
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Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8b). Brown powder; yield:
1
2
3%. H NMR (500 MHz, DMSOꢀd ) δ 7.59 (s, 1H), 5.74 (s, 2H), 4.23 (q, J = 7.1 Hz,
6
2H), 4.13 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
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
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48
49
50
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57
58
59
60
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀpropylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8c). Orange powder; yield:
1
6
6%. H NMR (500 MHz, DMSOꢀd
6
) δ 7.58 (s, 1H), 5.76 (s, 2H), 4.22 (q, J = 7.1 Hz,
2H), 4.06 (t, J = 7.1 Hz, 2H), 1.66ꢀ1.58 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H), 0.77 (t, J =
7.4 Hz, 3H).
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀisopropylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8d). White powder;
1
quantitative yield. H NMR (500 MHz, DMSOꢀd
6
) δ 7.73 (s, 1H), 5.73 (s, 2H), 5.09 (m,
1H), 4.23 (q, J = 7.1 Hz, 2H), 1.31 (d, J = 6.7 Hz, 6H), 1.27 (t, J = 7.1 Hz, 3H).
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀcyclopropylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8e). White powder;
1
yield: 85%. H NMR (500 MHz, DMSOꢀd ) δ 7.55 (s, 1H), 5.77 (s, 2H), 4.23 (q, J =
6
7.1 Hz, 2H), 3.62ꢀ3.56 (m, 1H), 1.27 (t, J = 7.1 Hz, 3H), 0.91ꢀ0.87 (m, 4H).
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀphenylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8f). Brown powder; yield:
1
7
7%. H NMR (500 MHz, DMSOꢀd
6
) δ 7.72 (s, 1H), 7.45ꢀ7.38 (m, 3H), 7.33ꢀ7.30 (m,
2H), 5.96 (s, 2H), 3.99 (q, J = 7.1 Hz, 2H), 0.95 (t, J = 7.1 Hz, 3H).
Ethylꢀ3ꢀaminoꢀ1ꢀbenzylꢀ4ꢀcyanoꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8g). Brown powder; yield:
1
7
1%. H NMR (500 MHz, DMSOꢀd ) δ 7.78 (s, 1H), 7.33ꢀ7.29 (m, 2H), 7.26ꢀ7.22 (m,
6
1H), 7.09ꢀ7.05 (m, 2H), 5.82 (s, 2H), 5.36 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 1.13 (t, J =
.1 Hz, 3H).
7
Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀphenethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8h). Brown powder;
1
yield: 90%. H NMR (500 MHz, DMSOꢀd ) δ 7.39 (s, 1H), 7.30ꢀ7.26 (m, 2H), 7.23ꢀ
6
7.18 (m, 1H), 7.15ꢀ7.12 (m, 2H), 5.78 (s, 2H), 4.33 (t, J = 7.4 Hz, 2H), 4.26 (q, J = 7.1
Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).
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Ethylꢀ3ꢀaminoꢀ4ꢀcyanoꢀ1ꢀ(3ꢀphenylpropyl)ꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate
(8i).
Brown
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powder; yield: 92%. H NMR (500 MHz, DMSOꢀd ) δ 7.59 (s, 1H), 7.28ꢀ7.24 (m, 2H),
6
7.19ꢀ7.15 (m, 3H), 5.78 (s, 2H), 4.19 (q, J = 7.1, 2H), 4.13 (t, J = 7.2 Hz, 2H), 2.52 (t,
J = 7.6 Hz, 2H), 1.97ꢀ1.89 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).
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Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀmethylꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylate (9a). Brown oil; quantitative yield. Used without further purification and
characterization.
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀethylꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylate (9b). Brown oil; quantitative yield. Used without further purification and
characterization.
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀpropylꢀ1Hꢀpyrroleꢀ2ꢀ
1
carboxylate (9c). Orange powder; quantitative yield. H NMR (500 MHz, DMSOꢀd ) δ
6
7.68 (s, 1H), 7.55 (s, 1H), 4.14 (t, J = 7.2 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 2.98 (s,
3H), 2.91 (s, 3H), 1.68ꢀ1.59 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H).
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀisopropylꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylate (9d). Brown oil; quantitative yield. Used without further purification and
characterization.
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ1ꢀcyclopropylꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylate (9e). Brown oil; quantitative yield. Used without further purification and
characterization.
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀphenylꢀ1Hꢀpyrroleꢀ2ꢀ
1
carboxylate (9f). Yellow needles; quantitative yield. H NMR (500 MHz, DMSOꢀd ) δ
6
7.82 (s, 1H), 7.74 (s, 1H), 7.47ꢀ7.38 (m, 3H), 7.32ꢀ7.28 (m, 2H), 3.92 (q, J = 7.1 Hz),
3.01 (s, 3H), 2.94 (s, 3H), 0.94 (t, J = 7.1 Hz, 3H).
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Ethylꢀ(E)ꢀ1ꢀbenzylꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1Hꢀpyrroleꢀ2ꢀ
1
carboxylate (9g). Brown powder; quantitative yield. H NMR (500 MHz, DMSOꢀd ) δ
6
7
5
3
.87 (s, 1H), 7.56 (s, 1H), 7.33ꢀ7.29 (m, 2H), 7.27ꢀ7.22 (m, 1H), 7.10ꢀ7.06 (m, 2H),
.46 (s, 2H), 4.03 (q, J = 7.1 Hz, 2H), 2.97 (s, 3H), 2.91 (s, 3H), 1.10 (t, J = 7.1 Hz,
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H).
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀphenethylꢀ1Hꢀpyrroleꢀ2ꢀ
1
carboxylate (9h). Orange powder; quantitative yield. H NMR (500 MHz, DMSOꢀd
6
) δ
7
4
2
.55 (s, 1H), 7.49 (s, 1H), 7.30ꢀ7.26 (m, 2H), 7.23ꢀ7.19 (m, 1H), 7.16ꢀ7.13 (m, 2H),
.40 (t, J = 7.5 Hz, 2H), 4.14 (q, J = 7.1 Hz), 2.99 (s, 3H), 2.94 (t, J = 7.7 Hz, 2H),
.92 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H).
Ethylꢀ(E)ꢀ4ꢀcyanoꢀ3ꢀ(((dimethylamino)methylene)amino)ꢀ1ꢀ(3ꢀphenylpropyl)ꢀ1Hꢀ
1
pyrroleꢀ2ꢀcarboxylate (9i). Orange powder; quantitative yield. H NMR (500 MHz,
DMSOꢀd ) δ 7.69 (s, 1H), 7.54 (s, 1H), 7.29ꢀ7.24 (m, 2H), 7.19ꢀ7.15 (m, 3H), 4.23 (t,
6
J = 7.1 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 2.98 (s, 3H), 2.91 (s, 3H), 2.53 (t, J = 7.6
Hz, 2H), 1.99ꢀ1.92 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H).
5ꢀmethylꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10a). White
1
powder; yield: 49%. H NMR (500 MHz, DMSOꢀd ) δ 12.31 (s, 1H), 8.14 (s, 1H), 7.94
6
(s, 1H), 4.01 (s, 3H).
5ꢀethylꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10b). Brown
1
powder; yield: 45%. H NMR (500 MHz, DMSOꢀd
6
) δ 12.34 (s, 1H), 8.25 (s, 1H), 7.95
(s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H).
4ꢀoxoꢀ5ꢀpropylꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10c). Brown
1
powder; yield: 24%. H NMR (500 MHz, DMSOꢀd ) δ 12.33 (s, 1H), 8.24 (s, 1H), 7.95
6
(s, 1H), 4.34 (t, J = 7.0 Hz, 2H), 1.81ꢀ1.74 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H).
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5ꢀisopropylꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10d). Brown
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powder; yield: 56%. H NMR (500 MHz, DMSOꢀd ) δ 12.26 (s, 1H), 8.41 (s, 1H), 7.96
6
(s, 1H), 5.30 (m, 1H), 1.44 (d, J = 6.7 Hz, 6H).
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5ꢀcyclopropylꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(10e).
1
Brown powder; yield: 6%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.13 (s, 1H), 7.94 (s, 1H),
4.10ꢀ4.04 (m, 1H), 1.09ꢀ0.98 (m, 4H).
4ꢀoxoꢀ5ꢀphenylꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10f). White
1
powder; yield: 21%. H NMR (500 MHz, DMSOꢀd ) δ 12.44 (s, 1H), 8.48 (s, 1H), 8.05
6
(s, 1H), 7.56ꢀ7.45 (m, 5H).
5ꢀbenzylꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10g). Yellow
1
powder; yield 47%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.31 (s, 1H), 7.97 (s, 1H), 7.35ꢀ
7.24 (m, 5H), 5.63 (s, 2H).
4ꢀoxoꢀ5ꢀphenethylꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(10h).
1
Yellow powder; yield: 43%. H NMR (500 MHz, DMSOꢀd
6
) δ 7.97 (s, 1H), 7.96 (s,
1H), 7.28ꢀ7.24 (m, 2H), 7.21ꢀ7.17 (m, 1H), 7.15ꢀ7.12 (m, 2H), 4.61 (t, J = 7.2), 3.08 (t,
J = 7.3, 2H).
4ꢀoxoꢀ5ꢀ(3ꢀphenylpropyl)ꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (10i).
1
White powder; yield: 21%. H NMR (500 MHz, DMSOꢀd ) δ 8.09 (s, 1H), 7.93 (s, 1H),
6
2
7.26ꢀ7.22 (m, 2H), 7.18ꢀ7.14 (m, 3H), 4.43 (t, J = 7.0, 2H, CH ), 2.54 (t, J = 7.7, 2H),
2.13ꢀ2.05 (m, 2H).
4ꢀchloroꢀ5ꢀmethylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11a). Brown powder;
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yield: 66%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.79 (s, 1H), 8.76 (s, 1H), 4.14 (s, 3H).
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4ꢀchloroꢀ5ꢀethylꢀ4,5ꢀdihydroꢀ3Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11b). Brown
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powder; yield: 79%. H NMR (500 MHz, DMSOꢀd ) δ 8.86 (s, 1H), 8.80 (s, 1H), 4.56
6
(q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H).
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ꢀchloroꢀ5ꢀpropylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11c). Brown powder;
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yield: 94%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.86 (s, 1H), 8.81 (s, 1H), 4.48 (t, J = 7.2
Hz, 2H), 1.88ꢀ1.81 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H).
4ꢀchloroꢀ5ꢀisopropylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11d). Brown powder;
1
yield 32%. H NMR (500 MHz, DMSOꢀd ) δ 9.05 (s, 1H), 8.80 (s, 1H), 5.40 (m, 1H),
6
1.54 (d, J = 6.6 Hz, 6H).
4ꢀchloroꢀ5ꢀcyclopropylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(11e).
Brown
1
powder; quantitative yield. H NMR (500 MHz, DMSOꢀd
6
) δ 8.83 (s, 1H), 8.80 (s, 1H),
3.93ꢀ3.88 (m, 1H), 1.28ꢀ1.14 (m, 4H).
4ꢀchloroꢀ5ꢀphenylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11f). Brown powder;
1
yield 94%. H NMR (500 MHz, DMSOꢀd
6
) δ 9.01 (s, 1H), 8.91 (s, 1H), 7.68ꢀ7.65 (m,
2H), 7.63ꢀ7.57 (m, 3H).
5ꢀbenzylꢀ4ꢀchloroꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11g). Brown powder;
1
yield 83%. H NMR (500 MHz, DMSOꢀd ) δ 8.98 (s, 1H), 8.83 (s, 1H), 7.36ꢀ7.32 (m,
6
2H), 7.31ꢀ7.27 (m, 1H), 7.16ꢀ 7.12 (m, 2H), 5.81 (s, 2H).
4ꢀchloroꢀ5ꢀphenethylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11h). Brown powder;
1
yield 88%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.81 (s, 1H), 8.65 (s, 1H), 7.28ꢀ7.24 (m,
2H), 7.21ꢀ7.19 (m, 1H), 7.15ꢀ7.11 (m, 2H), 4.77 (t, J = 7.4, 2H), 3.15 (t, J = 7.4, 2H).
4ꢀchloroꢀ5ꢀ(3ꢀphenylpropyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (11i). Brown
1
powder; yield 69%. H NMR (500 MHz, DMSOꢀd
6
) δ 8.84 (s, 1H), 8.79 (s, 1H), 7.26ꢀ
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(m, 2H).
General Procedure for the Preparation of Compounds 12-38. The title compounds
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were prepared as described in the literature with minor modifications. To a solution
of compounds 6a-b or 11a-i in 0.5 mL of dimethyl formamide a solution of the
corresponding primary or secondary amine in 0.5 mL of dimethyl formamide was
added. The reaction mixture was heated in the microwave (200 W, 110 °C, 20ꢀ60
minutes). The resulting product was purified by column chromatography with gradient
elution as described above. The products were crystallized in a mixture of ethanol
and petroleum ether (1:1).
4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀ
carbonitrile (12). Synthesized from 6a and phenylethylpiperazine; yield 66%, yellow
1
needles. H NMR (500 MHz, DMSOꢀd ) δ 8.46 (s, 1H), 7.29ꢀ7.22 (m, 4H), 7.19ꢀ7.15
6
(m, J = 7.1, 1.6 Hz, 1H), 3.34ꢀ3.29 (m, 4H), 4.35 (t, J = 5.6 Hz, 2H), 3.11 (t, J = 6.5
Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.61ꢀ2.68 (m, 4H), 2.58 (t, J = 7.4 Hz, 2H), 1.98ꢀ
13
1.87 (m, 4H). C NMR (126 MHz, DMSOꢀd
6
) δ 154.4, 151.2, 151.0, 150.2, 140.5,
1
3
28.8 (2C), 128.4 (2C), 126.0, 118.2, 114.4, 83.6, 59.8, 52.2 (2C), 50.4 (2C), 46.1,
+
2.84, 22.9, 22.6, 18.4. LCꢀMS: (m/z) calc.: 386.5; found: 387.4 [M+H] . Purity: 95%.
4ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
1
(
13). Synthesized from 6a and benzylpiperazine; yield 48%, yellow needles. H NMR
6
(500 MHz, DMSOꢀd ) δ 8.45 (s, 1H), 7.34ꢀ7.31 (m, 4H), 7.28ꢀ7.23 (m, 1H), 4.33 (t, J
= 5.6 Hz, 2H), 3.36ꢀ3.30 (m, 4H), 3.54 (s, 2H), 3.10 (t, J = 6.5 Hz, 2H), 2.66ꢀ2.51 (m,
1
3
4
1
4
6
H), 2.03ꢀ1.81 (m, 4H). C NMR (126 MHz, DMSOꢀd ) δ 154.3, 151.2, 150.9, 150.2,
38.1, 129.1 (2C), 128.3 (2C), 127.1, 118.2, 114.4, 83.6, 62.2, 52.3 (2C), 50.3 (2C),
+
6.1, 22.9, 22.6, 18.4. LCꢀMS: (m/z) calc.: 373.2; found: 372.5 [M+H] . Purity: 94%.
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4ꢀ(4ꢀbenzhydrylpiperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀ
carbonitrile (14). Synthesized from 6a and diphenylmethylpiperazine, yield 70%,
1
white needles. H NMR (500 MHz, DMSOꢀd
6
) δ 8.45 (s, 1H), 7.49ꢀ7.47 (m, 4H), 7.33ꢀ
7.27 (m, 4H), 7.21ꢀ7.16 (m, 2H), 4.37 (s, 1H, CH), 4.31 (t, 5.5 Hz, 2H), 3.30ꢀ3.40 (m,
10
11
12
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3
4
H), 3.09 (t, J = 6.5 Hz, 2H), 2.49ꢀ2.60 (m, 4H), 1.82ꢀ1.95 (m, 4H). C NMR (126
) δ 154.2, 151.3, 150.9, 150.13, 142.9 (2C), 128.7 (4C), 127.7 (4C),
27.1 (2C), 118.2, 114.4, 83.6, 75.4, 51.3 (2C), 50.4 (2C), 46,0, 22.9, 22.6, 18.4. LCꢀ
MHz, DMSOꢀd
6
1
+
MS: (m/z) calc.: 448.6; found: 449.4 [M+H] . Purity: 92%..
4
ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
1
(
15). Synthesized from 6a and phenylpiperazine; yield 13%, white needles. H NMR
6
(500 MHz, DMSOꢀd ) δ 8.50 (s, 1H), 7.24 (dd, J = 8.7, 7.3 Hz, 2H), 7.00 (d, J = 8.6
Hz, 2H), 6.81 (t, J = 7.3 Hz 1H), 4.40 (t, J = 5.6 Hz, 2H), 3.50ꢀ3.43 (m, 4H), 3.36ꢀ3.31
13
(
m, 4H), 3.13 (t, J = 6.4 Hz, 2H), 2.00ꢀ1.88 (m, 4H). C NMR (126 MHz DMSOꢀd ) δ
6
1
54.3, 151.3, 150.1, 150.1, 150.3, 129.1 (2C), 119.4, 118.4, 115.8 (2C), 114.4, 83.6,
50.3 (2C), 48.1 (2C), 46.1, 23.0, 22.6, 18.4. LCꢀMS: (m/z) calc.: 358.5; found: 359.4
+
[M+H] . Purity: 92%.
4
ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
1
(
(
(
16). Synthesized from 6a and methylpiperazine; yield 45%, white powder. H NMR
500 MHz, DMSOꢀd
6
) δ 8.45 (s, 1H), 4.34 (t, J = 5.6 Hz, 2H), 3.31ꢀ3.25 (m, 4H), 3.10
1
3
t, J = 6.5 Hz, 2H), 2.56ꢀ2.51 (m, 4H), 2.23 (s, 3H), 1.99ꢀ1.88 (m, 4H). C NMR (126
MHz DMSOꢀd
6
) δ 154.4, 151.2, 151.0, 150.2, 118.2, 114.5, 83.6, 54.2 (2C), 50.2
+
(
2C), 46.1, 45.8, 22.9, 22.6, 18.4. LCꢀMS: (m/z) calc.: 296.4; found: 297.2 [M+H] .
Purity: 100%..
4
ꢀ(piperazinꢀ1ꢀyl)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
(17).
1
Synthesized from 6a and piperazine; yield 59%, white powder. H NMR (500 MHz,
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DMSOꢀd
6
) δ 8.45 (s, 1H), 4.34 (t, J = 5.6 Hz, 2H), 3.23 (t, J = 4.6 Hz, 4H), 3.10 (t, J
13
6.5 Hz, 2H), 2.86 (J = 4.8 Hz, 4H), 1.98ꢀ1.86 (m, 4H). C NMR (126 MHz DMSOꢀd6)
δ 154.7, 151.3, 150.8, 150.2, 118.2, 114.5, 83.6, 51.6 (2C), 46.1, 45.2 (2C), 22.9,
+
22.6, 18.4. LCꢀMS: (m/z) calc.: 282.4; found: 283.2 [M+H] . Purity: 100%
0
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0
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4ꢀ((3ꢀphenylpropyl)amino)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
1
(18). Synthesized from 6a and phenylpropylamine; yield 40%, yellow neeldes. H
NMR (500 MHz, DMSOꢀd
6
) δ 8.20 (s, 1H), 7.25 (t, J = 7.5 Hz, 2H), 7.20 (d, J = 7.2
Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 6.80 (t, J 5.4 Hz, 1H), 4.37 (t, J = 6.1 Hz, 2H), 3.52ꢀ
3.47 (m, 2H), 2.99 (t, J = 6.3 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.03ꢀ1.98 (m, 2H),
13
6
1.95ꢀ1.89 (m, 2H), 1.86ꢀ1.80 (m, 2H). C NMR (126 MHz DMSOꢀd ) δ 152.2, 150.0,
147.7, 147.7, 142.1, 128.5 (2C), 128.5 (2C), 125.9, 115.1, 114.0, 82.8, 45.7, 33.0,
+
30.7, 23.1, 22.1, 18.2. LCꢀMS: (m/z) calc.: 331.4; found: 332.1 [M+H] . Purity: 99%.
4ꢀ(phenethylamino)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile (19).
1
Synthesized from 6a and phenylethylamine; yield 18%, yellow needles. H NMR (500
MHz, DMSOꢀd
6
) δ 8.24 (s, 1H), 7.29 (t, J = 7.4 Hz, 2H), 7.25 (d, J = 6.9 Hz, 2H), 7.20
(t, J = 7.3 Hz, 1H), 6.92 (t, J = 5.5 Hz, 1H), 4.34 (t, J = 6.1 Hz, 2H), 3.73ꢀ3.63 (m,
2
2
1
H), 3.00 (t, J = 6.3 Hz, 2H), 2.90 (t, J = 7.4, 2H), 2.04ꢀ1.97 (m, 2H), 1.97ꢀ1.80 (m,
13
H). C NMR (126 MHz DMSOꢀd ) δ 152.2, 149.7, 147.7, 147.6, 139.8, 128.8 (2C),
6
28.5 (2C), 126.2, 114.9, 114.0, 82.9, 45.6, 42.1, 35.1, 23.0, 22.0, 18.1. LCꢀMS:
+
(
m/z) calc.: 317.4; found: 318.1 [M+H] . Purity: 100%
4ꢀ(benzylamino)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
20).
1
Synthesized from 6a and benzylamine; yield 26%, yellow needles. H NMR (500
MHz, DMSOꢀd
6
) δ 8.17 (s, 1H), 7.46 (d, J = 6.4 Hz, 1H), 7.36 (d, J = 7.3 Hz, 2H),
7
6
.28 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H), 4.71 (d, J = 5.8 Hz, 2H), 4.47 (t, J =
13
.1 Hz, 2H), 3.01 (t, J = 6.3 Hz, 2H), 2.06ꢀ2.00 (m, 2H), 1.88ꢀ1.82 (m, 2H). C NMR
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(126 MHz DMSOꢀd
6
) δ 152.1, 149.6, 148.8, 147.8, 140.1, 128.3 (2C), 127.3 (2C),
126.7, 114.9, 113.9, 82.9, 45.8, 43.5, 23.1, 22.1, 18.2. LCꢀMS: (m/z) calc.: 303.4;
+
found: 304.1 [M+H] . Purity: 100%
10
11
12
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14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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60
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ꢀ(phenylamino)ꢀ6,7,8,9ꢀtetrahydropyrimido[4,5ꢀb]indolizineꢀ10ꢀcarbonitrile
(21).
1
Synthesized from 6a and aniline; yield 19%, yellow needles. H NMR (500 MHz,
6
DMSOꢀd ) δ 8.49 (s, 1H), 8.31 (s, 1H), 7.55 (d, J = 7.7 Hz, 2H), 7.33 (t, J = 7.9 Hz,
2H), 7.07 (t, J = 7.4 Hz, 1H), 4.57 (t, J = 6.1 Hz, 2H), 3.08 (t, J = 6.4 Hz, 2H), 2.07ꢀ
13
2
1
2
.02 (m, 2H), 1.90ꢀ1.85 (m, 2H). C NMR (126 MHz DMSOꢀd ) δ 151.5, 149.6,
6
49.2, 147.8, 139.6, 128.6 (2C), 123.4, 122.4 (2C), 114.9, 114.7, 83.22, 45.7, 23.2,
+
2.1, 18.3. LCꢀMS: (m/z) calc.: 289.3; found: 290.1 [M+H] . Purity: 98%
4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ7,8,9,10ꢀtetrahydroꢀ6Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀ
a]azepineꢀ11ꢀcarbonitrile (22). Synthesized from 6b and phenylethylpiperazine; yield
1
16%, yellow needles. H NMR (500 MHz, DMSOꢀd ) δ 8.47 (s, 1H), 7.29ꢀ7.22 (m,
6
4H), 7.19ꢀ7.15 (m, 1H), 4.59ꢀ4.39 (m, 2H), 3.40ꢀ3.28 (m, 4H), 3.14ꢀ3.03 (m, 2H), 2.77
(t, J = 7.2 Hz, 2H), 2.69ꢀ2.62 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 1.90ꢀ1.81 (m, 2H),
13
1.81ꢀ1.70 (m, 4H). C NMR (126 MHz, DMSOꢀd
6
) δ 156.6, 153.7, 151.0, 149.6,
1
4
40.5, 128.8 (2C), 128.3 (2C), 126.0, 117.9, 114.6, 85.4, 59.7, 52.1 (2C), 49.8 (2C),
+
7.4, 32.8, 29.9, 27.9, 27.1, 25.6. LCꢀMS: (m/z) calc.: 400.5; found: 401.3 [M+H] .
Purity: 96%
4ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ7,8,9,10ꢀtetrahydroꢀ6Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀ
a]azepineꢀ11ꢀcarbonitrile (23). Synthesized from 6b and benzylpiperazine; yield 36%,
1
white needles. H NMR (500 MHz, DMSOꢀd ) δ 8.46, (s, 1H), 7.35ꢀ7.31 (m, 4H),
6
7.28ꢀ7.22 (m, 1H), 4.52ꢀ4.40 (m, 2H), 3.36ꢀ3.28 (m, 4H), 3.54 (s, 2H), 3.12ꢀ3.04 (m,
13
2H), 2.69ꢀ2.51 (m, 4H), 1.87ꢀ1.79 (m, 2H), 1.80ꢀ1.67 (m, 4H). C NMR (126 MHz,
DMSOꢀd
25
6
) δ 156.6, 153.7, 151.1, 149.6, 138.1, 129.0 (2C), 128.4 (2C), 127.2, 117.9,
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114.6, 85.3, 62.1, 52.1 (2C), 49.7 (2C), 47.4, 27.9, 27.9, 27.1, 25.6. LCꢀMS: (m/z)
+
calc.: 386.5; found: 387.4 [M+H] . Purity: 99%
4ꢀ(4ꢀbenzhydrylpiperazinꢀ1ꢀyl)ꢀ7,8,9,10ꢀtetrahydroꢀ6Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀ
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a]azepineꢀ11ꢀcarbonitrile (24). Synthesized from 6b and diphenylmethylpiperazine;
1
yield 51%, white needles. H NMR (500 MHz, DMSOꢀd
6
) δ 8.46 (s, 1H), 7.48ꢀ7.43
(m, 4H), 7.32ꢀ7.27 (m, 4H), 7.21ꢀ7.16 (m, 2H), 4.49ꢀ4.39 (m, 2H), 4.38 (s, 1H), 3.40ꢀ
3.29 (m, 4H), 3.11ꢀ2.99 (m, 2H), 2.56ꢀ2.50 (m, 4H), 1.84ꢀ1.76 (m, 2H), 1.76ꢀ1.63 (m,
13
4
H). C NMR (126 MHz, DMSOꢀd ) δ 156.5, 153.6, 151.1, 149.8, 142.8 (2C), 128.7
6
(4C), 127.7 (4C), 127.1 (2C), 114.6, 117.9, 85.2, 75.2, 51.1 (2C), 49.8 (2C), 47.4,
+
2
9.9, 27.8, 27.1, 25.6. LCꢀMS: (m/z) calc.: 462.6; found: 463.3 [M+H] . Purity: 98%
4ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ7,8,9,10ꢀtetrahydroꢀ6Hꢀpyrimido[4',5':4,5]pyrrolo[1,2ꢀ
a]azepineꢀ11ꢀcarbonitrile (25). Synthesized from 6b and diphenylmethylpiperazine;
1
yield 52%, yellow needles. H NMR (500 MHz, DMSOꢀd ) δ 8.51 (s, 1H), 7.27ꢀ7.20
6
(m, 2H), 7.03ꢀ6.98 (m, 2H), 6.84ꢀ6.78 (m, 1H), 4.56ꢀ4.49 (m, 2H), 3.47ꢀ3.41 (m, 4H),
13
3
.38ꢀ3.31 (m, 4H), 3.14ꢀ3.09 (m, 2H), 1.90 ꢀ1.72 (m, 6H). C NMR (126 MHz,
) δ 156.8, 153.7, 151.1, 151.0, 149.7, 129.1 (2C), 119.4, 118.0, 115.9 (2C),
14.6, 85.3, 49.7 (2C), 47.9 (2C), 47.5, 29,9, 27.8, 27.1, 25.6. LCꢀMS: (m/z) calc.:
DMSOꢀd
6
1
3
+
72.5; found: 373.4 [M+H] . Purity: 99%
5ꢀmethylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (26).
1
Synthesized from 11a and phenethylpiperazine; yield: 72%, yellow needles. H NMR
6
(500 MHz, DMSOꢀd ) δ 8.50 (s, 1H), 8.44 (s, 1H), 7.29ꢀ7.25 (m, 2H), 7.25ꢀ7.22 (m,
2H), 7.19ꢀ7.15 (m, 1H), 4.01 (s, 3H), 3.41ꢀ3.34 (m, 4H), 2.77 (t, J = 7.3 Hz, 2H), 2.68ꢀ
13
2
.61 (m, 4H), 2.58 (t, J = 7.4 Hz, 2H). C NMR (126 MHz, DMSOꢀd
6
) δ 154.9, 151.3,
150.1, 141.5, 140.5, 128.8 (2C), 128.4 (2C), 126.0, 118.6, 114.4, 85.4, 59.8, 52.2
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+
(2C), 50.1 (2C), 36.8, 32.8. LCꢀMS: (m/z) calc.: 346.4; found: 347.2 [M+H] . Purity:
100%
5
ꢀethylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (27).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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46
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48
49
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58
59
60
1
Synthesized from 11b and phenethylpiperazine; yield: 64%, white needles. H NMR
(500 MHz, DMSOꢀd
6
) δ 8.61 (s, 1H), 8.54 (s, 1H), 7.29ꢀ7.22 (m, 4H), 7.19ꢀ7.15 (m,
1H), 4.34 (q, J = 7.2 Hz, 2H), 3.36ꢀ3.30 (m, 4H), 2.78 (t, J = 7.2 Hz, 2H), 2.70ꢀ2.62
13
(m, 4H), 2.59 (t, J = 7.3 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H). C NMR (126 MHz, DMSOꢀ
d6) δ 154.9, 151.4, 150.6, 140.6, 140.5, 128.8 (2C), 128.4 (2C), 126.0, 117.8, 114.3,
8
6.1, 59.7, 52.2 (2C), 50.1 (2C), 44.1, 32.8, 16.1. LCꢀMS: (m/z) calc.: 360.5; found:
+
3
61.2 [M+H] . Purity: 100%
4
ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5ꢀpropylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (28).
1
Synthesized from 11c and phenethylpiperazine; yield: 72%, white needles. H NMR
(
500 MHz, DMSOꢀd ) δ 8.59 (s, 1H), 8.55 (s, 1H), 7.29ꢀ7.22 (m, 4H), 7.20ꢀ7.15 (m,
6
1
H), 4.26 (t, J = 7.4 Hz, 2H), 3.33ꢀ3.30 (m, 4H), 2.78 (t, J = 7.2 Hz, 2H), 2.70ꢀ2.62 (m,
13
4
H), 2.59 (t, J = 7.3), 1.79ꢀ1.71 (m, 2H), 0.72 (t, J = 7.4 Hz, 3H). C NMR (126 MHz,
) δ 155.1, 141.4, 150.6, 141.2, 140.5, 128.8 (2C), 128.4 (2C), 126.0, 118.0,
14.3, 85.9, 59.7, 52.4, 50.8 (2C), 50.1 (2C), 32.8, 24.2, 10.7. LCꢀMS: (m/z) calc.:
DMSOꢀd
6
1
3
+
74.5; found: 375.2 [M+H] . Purity: 100%
5
ꢀisopropylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(
29). Synthesized from 11d and phenethylpiperazine; yield: 18%, pale yellow
1
needles. H NMR (500 MHz, DMSOꢀd
6
) δ 8.81 (s, 1H), 8.55 (s, 1H), 7.29ꢀ7.22 (m,
4H), 7.19ꢀ7.15 (m, 1H), 4.99 (hept, J = 6.7 Hz, 1H), 3.32ꢀ3.30 (m, 4H), 2.78 (t, J = 7.2
13
Hz, 2H), 2.70ꢀ2.61 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 1.44 (d, J = 6.6 Hz, 6H).
C
NMR (126 MHz, DMSOꢀd
6
) δ 154.9, 151.3, 150.3, 140.5, 138.3, 128.8 (2C), 128.4
(
27
2C), 126.0, 117.3, 114.4, 87.0, 59.7, 52.1 (2C), 50.0, 49.9 (2C), 32.8, 23.1 (2C).
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5ꢀcyclopropylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
1
(
30). Synthesized from 11e and phenethylpiperazine; yield: 47%, yellow needles. H
NMR (500 MHz, DMSOꢀd
6
) δ 8.46 (s, 1H), 8.45 (s, 1H), 7.29ꢀ7.21 (m, 4H), 7.19ꢀ7.15
(m,1H), 3.97 (quint, J = 3.8 Hz, 1H), 3.56ꢀ3.48 (m, 4H), 2.77 (t, J = 7.2 Hz, 2H), 2.64ꢀ
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
2
d
8
.60 (m, 4H), 2.57 (t, J = 7.4 Hz, 2H), 1.14ꢀ1.03 (m, 4H). C NMR (126 MHz, DMSOꢀ
6
) δ 154.4, 151.3, 150.3, 140.5, 140.2, 128.8 (2C), 128.4 (2C), 126.0, 118.6, 114.4,
5.7, 59.8, 52.4 (2C), 49.7 (2C), 32.8, 32.2, 8.5 (2C). LCꢀMS: (m/z) calc.: 372.5;
+
found: 373.3 [M+H] . Purity: 100%
4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5ꢀphenylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (31).
1
Synthesized from 11f and phenethylpiperazine; yield: 50%, yellow needles. H NMR
(500 MHz, DMSOꢀd
6
) δ 8.74 (s, 1H), 8.58 (s, 1H), 7.61ꢀ7.57 (m, 2H), 7.54ꢀ7.48 (m,
3
2
1
H), 7.25ꢀ7.21 (m, 2H), 7.17ꢀ7.12 (m, 3H), 3.12ꢀ3.00 (m, 4H), 2.61 (t, J = 7.4 Hz, 2H)
13
.34 (t, J = 7.5 Hz, 2H), 2.12ꢀ2.02 (m, 4H). C NMR (126 MHz, DMSOꢀd ) δ 153.2,
6
51.8, 151.2, 141.4, 140.4, 137.9, 129.6 (2C), 128.7 (2C), 128.5, 128.3 (2C), 125.9,
125.3 (2C), 115.3, 114.1, 88.2, 59.6, 51.4 (2C), 48.7 (2C), 32.5. LCꢀMS: (m/z) calc.:
+
408.5; found: 409.4 [M+H] . Purity: 100%
4
ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ5ꢀphenylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(32).
Synthesized from 11f and benzylpiperazine; yield: 15%, yellow needles. H NMR
500 MHz, DMSOꢀd ) δ 8.73 (s, 1H), 8.57 (s, 1H), 7.58ꢀ7.49 (m, 3H), 7.49ꢀ7.45 (m,
H), 7.30ꢀ7.25 (m, 2H), 7.25ꢀ7.21 (m, 1H), 719ꢀ7.16 (m, 2H), 3.31 (s, 2H), 312ꢀ3.00
1
(
6
2
13
(m, 4H), 2.04ꢀ1.91 (m, 4H). C NMR (126 MHz, DMSOꢀd
6
) δ 153.1, 151.8, 151.2,
1
1
3
41.4, 137.9, 137.7, 129.6 (2C), 128.9 (2C), 128.5, 128.3 (2C), 127.1, 125.3 (2C),
15.3, 114.1, 88.1, 61.9, 51.3 (2C), 48.7 (2C). LCꢀMS: (m/z) calc.: 394.5; found:
+
95.3 [M+H] . Purity: 100%
28
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Page 29 of 70
Journal of Medicinal Chemistry
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5ꢀbenzylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (33).
1
Synthesized from 11g and phenethylpiperazine; yield: 41%, yellow needles. H NMR
6
(500 MHz, DMSOꢀd ) δ 8.65 (s, 1H), 8.56 (1H), 7.33ꢀ7.11 (m, 10H), 5.52 (s, 2H),
3.39ꢀ3.30 (m, 4H), 2.76 (t, J = 7.2 Hz, 2H), 2.68ꢀ2.60 (m, 4H), 2.58 (t, J = 7.2 Hz, 2H).
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C NMR (126 MHz, DMSOꢀd ) δ 155.2, 151.7, 151.0, 141.7, 140.5, 137.0, 128.9
(2C), 128.8 (2C), 128.4 (2C), 128.1, 127.1 (2C), 126.0, 117.9, 114.1, 87.0, 59.7, 52.2
+
(2C), 52.1, 50.0 (2C), 32.8. LCꢀMS: (m/z) calc.: 422.5; found: 423.3 [M+H] . Purity:
1
00%
5
ꢀbenzylꢀ4ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
(34).
1
Synthesized from 11g and benzylpiperazine; yield: 13%, yellow solid. H NMR (500
MHz, DMSOꢀd
.51 (s, 2H), 3.54 (m, 2H), 3.36ꢀ3.30 (m, 4H), 2.57ꢀ2.52 (m, 4H). C NMR (126 MHz,
DMSOꢀd ) δ 155.1, 151.7, 151.0, 141.7, 138.0, 136.9, 129.1 (2C), 128.9 (2C), 128.3
6
) δ 8.64 (s, 1H), 8.55 (s, 1H), 7.35ꢀ7.22 (m, 8H), 7.13ꢀ7.09 (m, 2H),
13
5
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(2C), 128.1, 127.2, 127.1 (2C), 117.9, 114.0, 86.9, 62.1, 52.1 (2C), 52.1, 49.9. LCꢀ
+
MS: (m/z) calc.: 408.5; found: 409.4 [M+H] . Purity: 98%
5ꢀphenethylꢀ4ꢀ(4ꢀphenethylpiperazinꢀ1ꢀyl)ꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile
1
(35). Synthesized from 11h and phenethylpiperazine; yield: 60%, yellow needles. H
NMR (500 MHz, DMSOꢀd ) δ 8.55 (s, 1H), 8.49 (s, 1H), 730ꢀ7.21 (m, 4H), 7.20ꢀ7.09
6
(m, 4H), 7.00ꢀ6.96 (m, 2H), 4.55 (t, J = 7.1 Hz, 2H), 3.27ꢀ3.18 (m, 4H), 3.00 (t, J = 7.1
13
Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.69ꢀ2.60 (m, 4H), 2.58 (t, J = 7.3 Hz, 2H). C NMR
(126 MHz, DMSOꢀd ) δ 154.9, 151.3, 150.4, 141.1, 140.5, 137.3, 128.8 (2C), 128.7
2C), 128.4 (2C), 128.2 (2C), 126.7, 126.0, 118.1, 114.3, 86.2, 59.8, 52.2 (2C), 50.3,
6
(
+
5
0.0 (2C), 37.2, 32.7. LCꢀMS: (m/z) calc.: 436.6; found: 437.5 [M+H] . Purity: 100%.
4
ꢀ(4ꢀbenzylpiperazinꢀ1ꢀyl)ꢀ5ꢀphenethylꢀ5Hꢀpyrrolo[3,2ꢀd]pyrimidineꢀ7ꢀcarbonitrile (36).
1
Synthesized from 11h and benzylpiperazine; yield: 25%, white needles. H NMR (500
29
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