Synthesis and Biological Evaluation of N-[2-(4-Hydroxyphenylamino)-pyridin-3-yl]-4-methoxy-benzenesulfonamide (ABT-751) Tricyclic Analogues as Antimitotic and Antivascular Agents with Potent in Vivo Antitumor Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00847

Benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) were modified to produce tricyclic quinazolinone 15-18 or benzothiadiazine 26-27 derivatives. These compounds were evaluated in cytotoxicity and tubulin inhibition assays and led to potent inhibitors of tubulin polymerization. N-[2(4-Methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a) exhibited the best in vitro cytotoxic activity (GI₅₀ 10-66.9 nM) against the NCI 60 human tumor cell line and significant potency against tubulin assembly (IC₅₀ 0.812 μM). In mechanism studies, 16a was shown to block cell cycle in G2/M phase and to disrupt microtubule formation and displayed good antivascular properties as inhibition of cell migration, invasion, and endothelial tube formation. Compound 16a was evaluated in C57BL/6 mouse melanoma B16F10 xenograft model to validate its antitumor activity, in comparison with reference ABT-751 (1). Compound 16a displayed strong in vivo antitumor and antivascular activities at a dose of 5 mg/kg without obvious toxicity, whereas 1 needed a 10-fold higher concentration to reach similar effects.

Full text of DOI:10.1021/acs.jmedchem.6b00847

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Article
Synthesis and Biological Evaluation of N-[2-(4-Hydroxyphenylamino)-
pyridin-3-yl]-4-methoxy-benzenesulfonamide (ABT-751) Tricyclic Analogues
as Antimitotic and Antivascular Agents with Potent in Vivo Antitumor Activity.
Zacharie Segaoula, Julien Leclercq, Valerie Verones, Nathalie Flouquet, Marie Lecoeur, Lionel Ach,
Nicolas Renault, Amelie Barczyk, Patricia Melnyk, Pascal Berthelot, Xavier Thuru, and Nicolas Lebegue
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00847 • Publication Date (Web): 18 Aug 2016
Downloaded from http://pubs.acs.org on August 19, 2016
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Synthesis and Biological Evaluation of Nꢀ[2ꢀ(4ꢀ
Hydroxyphenylamino)ꢀpyridinꢀ3ꢀyl]ꢀ4ꢀmethoxyꢀ
benzenesulfonamide (ABTꢀ751) Tricyclic
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Analogues as Antimitotic and Antivascular Agents
with Potent in Vivo Antitumor Activity.
1
,2
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Zacharie Segaoula, Julien Leclercq, Valérie Verones, Nathalie Flouquet, Marie lecoeur,
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Lionel Ach, Nicolas Renault, Amélie Barczyk, Patricia Melnyk, Pascal Berthelot, Xavier
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Thuru, Nicolas Lebegue.
1
Univ. Lille, Inserm, CHU Lille, UMRꢀS 1172 ꢀ JPArc ꢀ Centre de Recherche JeanꢀPierre
AUBERT Neurosciences et Cancer, Fꢀ59000 Lille, France.
2
Oncovet Clinical Research, SIRIC ONCOLille, Parc Eurasante, Rue du Dr Alexandre Yersin, Fꢀ
59120 Loos, France.
3
Univ. Lille, CHU Lille, EA 7365 ꢀ GRITA ꢀ Groupe de Recherche sur les formes InJectables et
les Technologies Associées, Fꢀ59000 Lille, France.
4
Univ. Lille, Inserm, CHU Lille, U995 ꢀ LIRIC ꢀ Lille Inflammation Research International
Center, Fꢀ59000 Lille, France.
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KEYWORDS: Quinazolinone, benzothiadiazine, ABTꢀ751, tubulin polymerization, vascular
disrupting agent
ABSTRACT: Benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) were modified
to produce tricyclic quinazolinone 15ꢀ18 or benzothiadiazine 26ꢀ27 derivatives. These
compounds were evaluated in cytotoxicity and tubulin inhibition assays and led to potent
inhibitors of tubulin polymerization. Nꢀ[2(4ꢀMethoxyphenyl)ethyl]ꢀ1,2ꢀdihydroꢀpyrimidino[2,1ꢀ
b]quinazolinꢀ6ꢀone (16a), exhibited the best in vitro cytotoxic activity (GI 10–66.9 nM) against
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the NCI 60 human tumor cell line and significant potency against tubulin assembly (IC 0.812
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ꢁM). In mechanism studies, 16a was shown to block cell cycle in G2/M phase, to disrupt
microtubule formation, and displayed good antivascular properties as inhibition of cell
migration, invasion and endothelial tube formation. Compound 16a was evaluated in C57BL/6
mouse melanoma B16F10 xenograft model to validate its antitumor activity, in comparison with
reference ABTꢀ751 (1). Compound 16a displayed strong in vivo antitumor and antivascular
activities at a dose of 5 mg/kg without obvious toxicity, whereas 1 needed a 10ꢀfold higher
concentration to reach similar effects.
Introduction
Microtubules provide an important framework supporting cellular morphology in interphase,
they are the major components of the mitotic spindle which allows the controlled segregation of
the chromosomes during mitosis. Spindle poisons are described to interfere with the dynamic
instability of microtubules and to induce a cell cycle arrest in the G2/M phase, promoting a
1
mitotic catastrophe and finally causing cell death via apoptosis. Consequently, the microtubule
has become an important target for the design of new antimitotic anticancer agents. Amongst
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these antitumor agents, two maJor groups have been well characterized, microtubule stabilizers
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such as paclitaxel and microtubule destabilizers such as vinca alkaloids, colchicine,
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combretastatin Aꢀ4 (CAꢀ4), and sulfonamide ABTꢀ751 (1). Compound 1 is an orally
bioavailable sulfonamide molecule that interacts with the colchicine binding site on βꢀtubulin,
inhibiting microtubule polymerization. It exhibits a broadꢀspectrum antitumor activity in vitro in
a large panel of cancer cell lines including those showing resistance to other antimicrotubule
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agents. In addition, 1 displays antivascular activity, reducing tumorꢀassociated blood flow and
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thereby enhancing cytotoxic effects with little influence on normal vasculature. Compound 1
has shown excellent antitumor activity against a variety of xenograft models, such as nonꢀsmall
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cell lung cancer (NSCLC) and colon cancer.
As part of our program aiming at the identification of novel cytotoxic agents, a series of
benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) derivatives showing antiꢀ
tubulin polymerization activities, that could be considered as constraint analogs of 1, and with
potent in vitro and moderately in vivo antitumor activities have been earlier described (Figure
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1).
The relatively low in vivo antitumor activity of compound 2b1, has been explained by a
lack of chemical and metabolic stability while it displayed good oral bioavailability (F,%:
1
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54.2).
In order to improve the ADME properties, we have then extended our studies to the
synthesis of compounds bearing heterocyclic privileged structures which were important
scaffolds in medicinal chemistry and in agreement with the structureꢀactivity relationships
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previously established.
A beneficial approach in medicinal chemistry consists of exploration of privileged structures as
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new tolls in drug discovery.
Privileged scaffolds enhance the hit rates of therapeutic targets,
leading to compounds with improved drugꢀlike properties and could contribute to the discovery
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of other biologically targets of interest.
Quinazolinone is an heterocyclic core found in a
variety of biological active agents with a broad range of activities (antibacterial, antifungal,
antiviral, anticonvulsant, antitumor, sedative, antihypertensive…) that have been recently
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described.
Moreover, Quinazolinone skeleton has been demonstrated to generate antimitotic
agents, bearing their antitumor activity through the inhibition of the DNA repair processes or by
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the dysregulation of cell cycle progression of cancer cells.
Among the various classes of
antimitotics, quinazolinꢀ4ꢀones constitute an important part of wellꢀestablished
pharmacologically active compounds as they are associated with inhibitory effects on tubulin
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polymerization and the anticancer activities of 2ꢀstyrylquinazolinꢀ4ꢀones, 2ꢀaryl and 2,3ꢀ
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dihydroꢀ2ꢀarylquinazolinꢀ4ꢀones
and tetrahydropyrido[2,1ꢀb]quinazolinꢀ10ꢀones . From
these considerations, the quinazolinone skeleton could be considered as a privileged structure.
Therefore, this paper describes the synthesis of five or sixꢀmembered rings containing nitrogen
atoms based on 2,3ꢀfused quinazolinꢀ4ꢀone systems (15ꢀ18) and their sulfonyl benzothiadiazine
analogues (26ꢀ27) as novel, highly potent tubulin polymerization inhibitors.
Results and Discussion
Chemistry. The synthetic route to 2,3ꢀfused quinazolinꢀ4ꢀone derivatives 15ꢀ18 is shown in
Scheme 1. The 2ꢀchloroꢀ3Hꢀquinazolinꢀ4ꢀone key intermediate 6 was synthesized by using the
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general procedure described by Hess and coꢀworkers. Commercial available anthranilamide 3
was first treated with potassium cyanate to yield quinazolineꢀ2,4ꢀdione 4 which reacted with
POCl in the presence of N,Nꢀdimethylaniline to provide 2,4ꢀdichloroquinazoline 5 in 88% yield.
3
Selective hydrolysis of 5 with 2% aqueous sodium hydroxide solution at 30°C gave the 2ꢀchloroꢀ
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Hꢀquinazolinꢀ4ꢀone 6 in 83% yield. Displacement of the chlorine with amino acetaldehyde or 3ꢀ
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aminopropionaldehyde diethyl acetal followed by treatment with concentrated sulfuric acid
allowed the linear cyclisation to 1Hꢀimidazo[2,1ꢀb]quinazolinꢀ5ꢀone 9 and 1,2ꢀdihydroꢀ1Hꢀ
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pyrimidino[2,1ꢀb]quinazolinꢀ6ꢀone 10 with respectively 60% and 89% yield. Arylethyl
methanesulfonates 14aꢀd were obtained in three steps from the corresponding benzaldehydes by
first, Wittig olefination with methyltriphenylphosphonium iodide followed by hydroboration
leading to primary alcohols 13aꢀd which finally reacted with methanesulfonyl chloride.
Treatment of compounds 9 and 10 with sodium hydride in anhydrous DMF followed by addition
of the arylethyl methanesulfonates 14aꢀd provided the corresponding alkylated derivatives 15aꢀd
and 16aꢀd in low yields resulting in a competition between substitution and elimination
reactions. Hydrogenation of the double bond but also hydrogenolysis of the benzyl protection of
compounds 15d and 16d was carried out by decomposition of ammonium formate in the
presence of palladium catalyst in ethanol leading to compounds 17aꢀd and 18aꢀd in moderate
yields. Access to unsaturated ꢀphenol derivatives 15e and 16e was performed by cleavage of the
benzyl ether group in strong acid conditions of the acidꢀinsensitive substrates 15d and 16d.
The benzothiadiazine analogues 26 and 27 were synthesized following the general procedures
as detailed below (Scheme 2). 2ꢀMethylthioꢀimidazolidine hydriodide 20 or 2ꢀbenzylthioꢀ
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1,4,5,6ꢀtetrahydropyrimidine hydriodide 21, obtained from the reaction of the iodide salts of 2ꢀ
imidazolidinethione or tetrahydroꢀ2ꢀpyrimidinethione with respectively methyl iodide or benzyl
bromide, were reacted with 2ꢀnitrobenzenesulfonyl chloride to give the corresponding
sulfonamides 22 and 23. Reduction with zinc in acidic conditions of the nitro group followed by
a simultaneous nucleophilic displacement of alkylthio group was described to conduct to the
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intramolecular cyclization with the resulting hydroxylamino group as maJor compound.
A
modification of this previously procedure using tin (II) chloride in refluxing ethanol with a
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catalytic amount of hydrochloric acid caused the exclusive formation of the deoxygenated
benzothiadiazines 24 and 25. Condensation of these tricyclic heterocycles with arylethyl
methanesulfonates 14aꢀd and hydrogenolysis of the benzyl protections were performed as
described in scheme 1 and provided the target compounds 26ꢀ27.
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We planned to synthesize the corresponding unsaturated derivatives 28ꢀ29 by a key step of
oxidative dehydrogenation using Pd/C in refluxing xylene but this reaction led only to recovery
of starting material. We then decided to use DDQ as aromatization reagent to effect facile
dehydrogenation of both simple and complex hydroaromatic heterocyclic compounds.
Compounds 26a was then refluxed in toluene with 2 eq. of DDQ and not conducted to the
desired unsaturated derivative 28a but to single product which proved to be the cyclocondensed
imidazobenzothiadiazines 30 bearing a chloro substituent and was isolated in only 25% yield.
Several substrates were then prepared and described recently to explore the scope of this
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reaction.
Biological Studies. Antiproliferative Activity in Cellular Assays. The in vitro cytotoxicity of
all synthesized compounds against human colon adenocarcinoma HT29 cell line was
investigated, in parallel with compound 2b1 and 1 as positive references, by the MTS assay after
the incubation of cells with the compounds for 72 h. Data are expressed as percentage of cell
proliferation inhibition at a high single dose (10 µM) or IC defined as the concentration at
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which the compounds reduce cell proliferation by 50% and are shown in Table 1. The results
indicate that inhibition of cell proliferation was highly dependent upon the size of the fused ring
and the presence of the double bond. Fiveꢀmembered rings were less active than their sixꢀ
membered counterparts in which the presence of a double bond enhanced the antiproliferative
activity. Indeed, compounds bearing a dihydroꢀimidazole as fused ring were not (17aꢀc) or
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poorly (17d) active while introduction of a double bond improved antiproliferative activities with
IC50 values in the micromolar range (15a,e). Increasing the ring size by introduction of a
tetrahydroꢀpyrimidine as sixꢀmembered ring led to better compounds (e.g., 18a,b vs 17a,b) in
which the presence of a double bond enhanced the antiproliferative activity from the
submicromolar to the nanomolar potency (e.g., 18a vs 16a). Replacement of the carbonyl group
by its sulfonyl bioisostere generated a general decrease of the cytotoxicity as for example, the
sulfonyl analogue 27a which was fiveꢀfold less potent than its direct carbonyl counterpart 16a.
Concerning the arylethyl side chain, the number and position of hydroxyl or methoxy
substituents on the phenyl ring had a maJor influence on antiproliferative properties. The
presence of a 3ꢀhydroxyꢀ4ꢀmethoxyphenyl substitution in both fiveꢀ and sixꢀmembered ring
series provided good antiproliferative activities at the micromolar range (15e, 16e, 17d, 18d) but
the greatest activities occurred in the presence of a 4ꢀmethoxyphenyl substitution in the sixꢀ
membered series (16a, 18a). Introduction of a 3,4ꢀdimethoxyꢀ or 3,4ꢀ5ꢀtrimethoxyphenyl group,
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a typical pharmacophore found in many inhibitors of tubulin polymerization, led to less active
compounds (15b, 16b, 18b) or a total loss of antiproliferative activity (15c, 16c, 17c, 18c).
These results allowed to identify four new active antiproliferative compounds (16a, 16b, 18a
and 27a) displaying low IC values ranging from 42 to 766 nM (Table 1). Structureꢀactivity
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tolerant of heterocyclic cores, (2) replacement of the carbonyl group by a sulfonyl moiety did not
led to positive results, and (3) the 4ꢀmethoxy substitution on the phenylethyl side chain is more
favorable than a 3ꢀhydroxyꢀ4ꢀmethoxy substitution.
NCI's in vitro 60 cell screening. Compounds 16a, 18a and 27a were selected by the National
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tested at a high single dose (10 µM) in the full 60ꢀcell panel and have progressed to the 5ꢀdose
screen in order to evaluate their GI values (Table 2). A mean graph midpoints (MGꢀMID) was
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over all cell lines for the tested compounds. These data indicated that quinazolinone 16a, with an
average GI of 58.9 nM, was more active than its saturated analogue 18a (GI (MGꢀMID):
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99.5 nM) and benzothiadiazine 27a (GI (MGꢀMID): 645 nM) which displayed weak growth
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inhibition potentials in several cancer cell lines as NSCL (NCIꢀH226), melanoma (UACCꢀ62),
ovarian (IGROV1, SKꢀOVꢀ3) or renal (UOꢀ31) cancers. Exhibiting the best activities
comparable to our previously highlighted benzopyridooxathiazepine (2), compound 16a was
then selected for further biological evaluations.
Cell Cycle Analysis. The effects of 16a, the most active compound on cell division process
were evaluated using SKꢀMel 28 melanoma cell line. The product was applied at varying
concentrations, between 100 nM and 10 µM for 24h in parallel with vehicle (1% DMSO). The
cells were then stained with propidium iodide (PI) and analyzed by flow cytometry. As
represented in figure 2, cell populations treated with 16a were significantly arrested with an
accumulation in the G2/M phase and an increase in subG1 apoptotic cells in a dose dependent
manner and it was accompanied by a comparable reduction in the proportion of cells in both the
G1 and S phases of the cell cycle. No effects on G2/M cell cycle blockade have been reported for
the control populations which remained in a G0/ G1 state mainly. These results clearly indicated
that the cell cycle arrest caused by 16a, as evidenced by the ability of the incubated cells to pass
through the G2/M phase, was generally observed for antimitotic drugs that bind to tubulin.
Immunocytochemical analysis. To determine the microtubule disrupting effects of the target
compounds, we selected 16a as a representative compound in a cellꢀbased phenotypic screening
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(
Figure 3). SKꢀMel 28 cells were used to examine the effect of 16a on the reorganization of
microtubules during mitosis. Cells were treated for 6h with 16a or vehicle (1% DMSO), and
microtubules were visualized by indirect immunofluorescence techniques. An antibody for βꢀ
tubulin was used to visualize interphase and mitotic microtubule structures. Vehicle treated cells
displayed a spider’s web like network of fibrous filaments (green) wrapped around the cell
nucleus (blue) (Figure 3A,B,C). Morphological aspects of untreated vs. 16a treated cells showed
a significant difference in the microtubules structure between the two populations and a loss of
polarity of 16a treated SKꢀMel 28 cells which adopted a spherical configuration due to the
disorganization of the microtubule network (Figure 3D,F).
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Tubulin content. In order to evaluate whether our compounds affected polymerization or
depolymerization of tubulin, an immunoblot analysis on protein extracts was performed to
determine the polymerized and soluble tubulin fractions. Cells were treated for six hours with
compound 16a (1 µM) or vehicle. The results showed a significant decrease in polymerized
tubulin fractions between 16a treated group and the related controls (untreated, vehicle) (36% ±
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vs. 100% ± 20, p value=0.0071, n=4) whereas no significant change have been reported in the
soluble fractions between all groups (Figure. 4). Thus, these data confirmed that compound 16a
induced its antimitotic effects by inhibiting tubulin polymerization since s/p tubulin ratio
increased by a fold of two in 16a treated cells indicating more soluble protein form.
Inhibition of Tubulin Polymerization. Compounds 15ꢀ18 were then evaluated for their in vitro
inhibition of tubulin polymerization in comparison with compound 2b1 and 1 (Table 1). In this
assembly assay, the sixꢀmembered ring derivatives 16a,b,e, 18a, 27a,e displayed greater
inhibitory activities than the reference compounds 2b1 and 1, with IC values ranging from 0.27
5
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to 1.29 µM whereas the others seemed to be inactive as inhibitors of tubulin polymerization and
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did not inhibit tubulin assembly at concentrations as high as 10 µM. Inhibitions of tubulin
polymerization of tested compounds were consistent with their antiproliferative activities.
Indeed, compounds bearing a 3,4,5ꢀtrimethoxy group were inactive at concentrations as high as
0
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10 µM while other variations of substituents within the aryl moiety as 4ꢀmethoxy (16a, 18a,
27a), 3,4ꢀdimethoxy (16b) and 3ꢀhydroxyꢀ4ꢀmethoxy groups (16e, 27e), led to significant
activities with higher potencies than the reference compounds. In contrast with the cell
proliferation assays, the carbonyl derivatives 16a,e were less potent than their counterparts 27a,e
whose substitution with a sulfonyl group seemed to be most tolerant for tubulin polymerization
inhibitory effect. Among all compounds tested, 16a, 18a and 27a were identified as the most
active ones in both cell proliferation and tubulin assembly assays. The discrepancy between
cytotoxicity and antitubulin activity of compounds 16e and 27e can be explained by the high
resistance of HT29 cell line to most of tubulin polymerization inhibitors with a 3ꢀhydroxyꢀ4ꢀ
3
1
methoxy substitution as for CAꢀ4. It has been reported that glucuronidation of phenol
derivatives as CAꢀ4 is highly observed in HT29 colorectal cancer cells due to an overexpression
32
of glucuronosyl transferase activity and then contributes to drug resistance phenomenon.
Molecular Modeling. Molecular docking tools are used for the structureꢀbased analysis in
order to elucidate the interactions of best compounds 16a and 27a of each class of quinazolinone
and benzothiadiazine derivatives. The structural similarity between reference compound 1 and
16a and 27a made consistent the docking of these two new molecules into the cavity of the 1ꢀ
33
34
bound tubulin crystal structure (PDB ID 3HKC). Using AutodockVina with a 50 Å edged
cubic box including one βꢀtubulin subunit and centered onto compound 1 binding site allowed to
retrieve the first ranked docking pose of each compound very close to the coꢀcrystallized
compound 1 (Figure 5) suggesting they could be really considered as constraint analogs of 1.
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29,35
According to previous studies,
the main receptorꢀligand interactions were hydrophobic
contacts with Leu240, Leu255, Ala314, Val316 and Ile378 of β chain. In comparison with
compound 1, an additional interaction was observed between carbonyl of 16a and sulfonyl of
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2
7a and thiol group of Cys239 side chain. Interestingly, this amino acid was shown to be
involved in the conformational changes of the various transition states during tubulin
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polymerization. Arguing for a prediction of inhibition of polymerization by alpha/beta tubulin
disruption rather than the prediction of cellular antiꢀproliferative activity, it emphasizes an
inhibition of polymerization (0,812 ꢁM and 0,27 ꢁM) in the same order of magnitude by
compounds 16a and 27a. By keeping suitable enthalpic contributions in term of intermolecular
specificity and decreasing entropic contribution by a strategy of rigidification of compound 1,
these docking results support the original design aiming at increase the potency of constraint
analogs of 1.
HUVEC Cell invasion and endothelial tube formation assays. To continue its growth and
expansion, a neoplastic tissue requires oxygen and metabolic supplies, that’s why, the tumor
microenvironment stimulates the development of additional blood vessels from already
3
8
preexistent branches; this process is well known under the term of angiogenesis. Recent
antitumor strategies are based on the use of chemotherapeutic agents with antiangiogenic or
antivascular drugs in order to increase the efficacy of the treatment. Besides the ability to inhibit
tumor cell proliferation, microtubuleꢀtargeting drugs have also been shown to be active against
39,40
the tumor vasculature by inhibiting endothelial cell invasion and capillary tube formation.
Considering that tube formation and invasion are highly relevant properties in the process of
tumor vasculature, we investigated the effects of compound 16a and antiangiogenic inhibitor
41
cediranib (31, VEGFR, PDGFR and cꢀKit inhibitor) , as the reference substance at several
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concentrations (0.001, 0.01, 0.1 and 1 µM) on the invasiveness of human umbilical vein cord
endothelial cells (HUVEC) in a Boyden chamber assay. Transwell instets were precoated with
basement membrane matrix and the number of HUVEC that penetrated the membrane was
quantified. As shown in Figure 6, treatment with compound 16a inhibited HUVEC cell invasion
at nontoxic concentrations lower than 0.01 µM ( 74% ±0.01 inhibition, p value < 0.001) while 31
displayed similar effects at a more pronounced concentration of 1 µM ( 74% ±0.09, p value <
0
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0.001). We also studied the effects of compound 16a on vascular tube formation. The
antiangiogenic effect was evaluated on HUVEC model by treating the cells with either the
reference compound 31 or compound 16a. Cells were seeded on a specific membrane matrix,
which is rich in proꢀangiogenic factors that stimulate single endothelial cells to assume an
extended shape, in order to mimic in vivo HUVEC and treated with several concentrations of 31
or 16a (0.001, 0.01, 0.1 and 1 µM). The overall effect results in a reticular similar to a capillary
4
1
network and offers an in vitro model of angiogenesis. As shown in Figure 7, compound 16a
significantly displayed vascular disrupting effects at 0.1 ꢁM with total destruction of the cordlike
structures at 1 ꢁM and at which 31 was less effective.
SK-Mel28 cell wound healing assay. The effect of compound 16a on cell migration, which is
an important mechanism involved in tumor invasion, was also evaluated by scratching a
melanoma SKꢀMel28 cell line monolayer and monitoring the percentage of wound closure. As
shown in Figure 8, compound 16a was very efficient in arresting cell motility. The effect was
statistically significant after 9 h of incubation at the concentration of 1µM, in comparison with
control cells, keeping the wound nearly intact (wound closure 3% ± 2, p value < 0.001).
Druglike properties. The hydrophobic character (logP) and the in vitro metabolic stability of
16a lead compound have been determined and compared with those of the reference compound
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b1. Quinazolinone 16a exhibited lower logP value (2.63 ± 0.01 versus 3.10 ± 0.01) and greater
metabolic stability in rat liver microsome (t1/2 = 56 min versus 38 min) than
benzopyridooxathiazepine derivative 2b1. The determination of MichaelisꢀMenten parameter
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(
K ), which is reciprocally proportional to the enzyme affinity to the substrate, also confirmed
m
the previous results with K values of 5.2 ± 1.0 µM and 27.2 ± 5.6 µM for compounds 2b1 and
m
1
6a, respectively. Its improved druglike properties as well as its antiproliferative and
antivascular activities suggest that compound 16a may be a better potential drug candidate than
its oxathiazepine analogue 2b1.
Antitumor Activitiy In Vivo. To further investigate the in vivo effects of compound 16a on
4
2
tumor growth and vascularization, we used a syngeneic melanoma mouse model (B16F10). In
in vitro preliminary experiments, we demonstrated that compound 16a showed potent cytotoxic
activity against melanoma cell lines. C57BL/ 6 mice were then subcutaneously inJected with
B16F10 murine melanoma cell lines and when tumor bulging reached a measurable size (about
3
1
00 mm ); mice were divided and randomly assigned to six groups. Three different doses
ranging from 5 mg/ kg /day to 50 mg/ kg /day were tested for 16a compared to a single 50 mg/
4
3
kg /day dose for reference 1. Treatment was delivered continuously using subcutaneous
osmotic pumps and in accordance with the pharmacokinetic data. As depicted in figure 9 and 10,
product 16a showed a significant decrease in tumor growth and angiogenesis at the starting 5
3
3
mg/ kg dose as compared with the administration of vehicle (1000mm ± 280 vs. 2400mm ±
10, p value = 0.009) and a 60% decrease in the percentage of microvessel number with no
3
significance to the standard compound 1 treatment (p value <0.005). At the lower dose, no
significant difference was observed between 16a and 1 on tumor growth inhibition and
angiogenesis, but a slight difference was shown at the higher 50 mg/ kg/ day with no statistical
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significance (760mm ± 160 vs. 1200mm ± 390, p value = 0.25 and 80% decrease in
3
microvessel number) in comparison to vehicle (2400mm ± 310, p value = 0.0007). Mice
monitoring showed no significant difference in mice average weight between all groups before,
during and after treatment initiation. Hence, compound 16a showed strong antitumor and
antivascular activities on a wellꢀtolerated dose schedule and was as effective as 10ꢀfold lower
dose of reference compound 1.
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Conclusion
By structural modifications on the thiadiazepine and oxathiazepine moieties in prior leads 1
and 2, a series of 2,3ꢀfused quinazolinꢀ4ꢀone systems (15ꢀ18) and their sulfonyl benzothiadiazine
analogues (26ꢀ27) were synthesized and evaluated in cytotoxicity and tubulin inhibition assays
and led to a new class of inhibitors of tubulin polymerization. The most potent compound, 16a
exhibited the best in vitro cytotoxic activity in cellular assays with a mean GI value of 58.9 nM
50
on the NCI60 human tumour cell line and significant potency against tubulin assembly with an
IC value of 0.812 ꢁM. In mechanism studies, 16a was shown to block cell cycle in G2/M
5
0
phase, inhibited cell invasion and migration and displayed antiangiogenic effect, thus providing
evidence that this tubulin polymerization inhibitor acts as vascular disrupting agents (VDAs).
SAR and molecular modeling studies suggested that the presence of a carbonyl or a sulfonyl
group allowed additional interactions with thiol group of Cys239 side chain which might be
responsible for the greater potency of 16a and 27a than the reference 1. Following druglike
property assessments, 16a was evaluated in parallel with reference 1 in C57BL/6 mouse
melanoma B16F10 xenograft models to evaluate their in vivo activities. Compound 16a
exhibited strong in vivo antitumor and antivascular activities, suppressing tumor growth and
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angiogenesis equally to 1 but at a 10ꢀfold lower dose. In summary, these newly tricyclic
quinazolinone or benzothiathiazine derivatives displayed marked biological activities in vitro and
in vivo as a novel class of anticancer agents and have potential for further development.
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Experimental Section
1. General Chemistry. All reagents and solvents were purchased and used without further
purification. Melting points were determined on a BÜCHI Bꢀ540 apparatus and are uncorrected.
1
NMR spectra were recorded on a Bruker Avance 300 spectrometer operating at 300MHz ( H) or
13
7
5MHz ( C). Chemical shifts are in parts per million (ppm) and were referenced to the residual
proton peaks in deuterated solvents. Mass spectra were recorded with an LCMS (Waters Alliance
Micromass ZQ 2000). LCMS analysis was performed using a Waters XBridge C18 column (5
µm particle size column, dimensions 50 mm x 4.6 mm). A gradient starting from 98%
H O/formate buffer 5 mM (pH 3.8) and reaching 100% CH CN/ formate buffer 5 mM (pH 3.8)
2
3
within 4 min at a flow rate of 2 mL/min was used followed by a return to the starting conditions
within 1 min. Purities of tested compounds reached at least 95% and were determined by HPLC
using the following instruments and conditions: Waters HPLC system was equipped with a
HPLC 600 pump and a 2996 photodiode array detector. The HPLC analysis were performed on a
LiChroprep Hibar® column (25ꢀ40 µm particle size column, dimensions 250 mm x 4.6 mm),
detection at 254 nm, and injection volume of 20 ꢁL. Mobile elution was conducted with a
mixture of petroleum ether (PE):ethyl acetate (EA) (see sup info).
Procedure for synthesis of compound 4. Anthranilic acid 3 (15 g, 0.109 mol) was suspended
in water (500 mL) and glacial acetic acid (6.75 mL) at 35 °C. A freshly prepared solution of
potassium cyanate (11.5 g, 0.142 mol) in water (100 mL) was added dropwise to the stirred
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mixture. After 2 h, sodium hydroxide (144 g 3.6 mol) was added in portions, keeping the
temperature below 40 °C. A clear solution was obtained momentarily before precipitation of the
hydrated sodium salt. After cooling, the precipitate was filtered off and dissolved in hot water
then acidified to pH 5, causing precipitation of 1Hꢀquinazolineꢀ2,4ꢀdione which was filtered and
dried.
0
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7
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1
H-quinazoline-2,4-dione (4). Yield 74%, 13 g. White solid, mp 360ꢀ362 °C (Lit. 355ꢀ
1
4
3
3
58°C). H NMR (DMSOꢀd , 300 Hz): 7.15 (2H, m, CH quina), 7.62 (1H, td, J = 1.5 Hz and J
6
4
3
= 7.3 Hz, CH quina), 7.88 (1H, dd, J = 1.5 Hz and J = 8.2 Hz, CH quina), 11.2 (2H, s, NH).
C H N O MW= 162.14 g/mol.
8
6
2
2;
Procedure for synthesis of compound 5. A mixture of 1Hꢀquinazolineꢀ2,4ꢀdione (12 g, 0.074
mol), trichlorophosphate (41.5 mL, 0.445 mol) and N,Nꢀdimethylaniline (6.1 mL, 0.048 mol) is
refluxed for 5h. The excess of trichlorophosphate is evaporated and ice is added to the residue.
After standing at room temperature overnight, the mixture was filtered to give a solid which was
recrystallized from methanol.
45
2
,4-dichloroquinazoline (5). Yield 88%, 12.95 g. Offꢀwhite solid, mp 119ꢀ121 °C (Lit. 118ꢀ
1
4
3
120°C). H NMR (DMSOꢀd , 300 Hz): 7.91 (1H, td, J = 1.2 Hz and J = 6.9 Hz, CH quina),
6
4
3
4
3
8
.05 (1H, dd, J = 1.2 Hz and J = 8.3 Hz, CH quina), 8.18 (1H, td, J = 1.5 and J = 6.9 Hz, CH
4
3
quina), 8.31 (1H, dd, J = 1.5 Hz and J = 8.5 Hz, CH quina). C H N Cl ; MW= 199.03 g/mol
.
8
4
2
2
Procedure for synthesis of compound 6. A suspension of 2,4ꢀdichloroquinazoline (2.8 g, 14.1
mmol) was stirred in 2% aqueous sodium hydroxide solution (50 mL) for 24 h at 30 °C. The
reaction mixture was diluted with water (100 mL) and filtered to remove unreacted 2,4ꢀ
dichloroquinazoline. The filtrate was neutralized with dilute acetic acid, the precipitate thus
obtained was filtered and washed with water.
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2
-chloro-3H-quinazolin-4-one (6). Yield 83%, 2.1 g. Offꢀwhite solid, mp 218ꢀ220 °C (Lit.
1
4
2
19ꢀ221 °C). H NMR (DMSOꢀd , 300 Hz): 7.57 (2H,m, 2H, CH quina), 7.83 (1H, td, J = 1.8
6
3
4
3
Hz and J = 7.3 Hz, CH quina), 8.09 (1H, dd, J = 1.8 Hz and J = 7.7 Hz, CH quina), 13.25 (1H,
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s, NH). C H ClN O MW= 180.59 g/mol.
8
5
2
;
General Procedure for synthesis of compounds 7 and 8. A mixture of 6 (1.8g, 0.01 mol) and
2,2ꢀdiéthoxyethylamine (4.35 mL, 0.03 mmol) or 3,3ꢀdiethoxypropylamine (4.85 mL, 0.03 mol)
is refluxed in ethanol (35 mL) for 4h. After cooling, the reaction mixture is filtered and the
filtrate evaporated under vacuum. The residue is precipitated in water, filtered and recrystallized
from ethanol.
2-(2,2-diéthoxyéthylamino)-3H-quinazolin-4-one (7). Yield 71%, 2.0 g. Yellow solid, mp 176ꢀ
2
6
1
3
178 °C (Lit. 174ꢀ180°C). H NMR (DMSOꢀd , 300 Hz): 1.12 (6H, t, J = 7.5 Hz, CH ), 3.44
6
3
3
3
(
2H, t, J = 5.5 Hz, CH ), 3.51 (2H, m, OCH ), 3.67 (2H, m, OCH ), 4.64 (1H, t, J = 5.5 Hz,
2 2 2
3
3
CH), 6.17 (1H, s, NH), 7.10 (1H, t, J = 7.8 Hz, CH quina), 7.22 (1H, d, J = 8.65 Hz, CH quina),
3
3
7
.56 (1H, t, J = 7.8 Hz, CH quina), 7.87 (1H, d, J = 7.8 Hz, CH quina), 10.80 (1H, s, CONH).
C H N O ; MW= 277.31 g/mol.
14
19
3
3
2-(3,3-diéthoxypropylamino)-3H-quinazolin-4-one (8). Yield 88%, 2.55 g. Yellow solid, mp
1
3
9
9ꢀ101 °C. H NMR (DMSOꢀd , 300 Hz): 1.15 (6H, t, J = 7.5 Hz, CH ), 1.72 (2H, m, CH ),
6 3 2
3
3
.33 (2H, m, NCH ), 3.46 (2H, m, OCH ), 3.65 (2H, m, OCH ), 4.59 (1H, t, J = 5.5 Hz, CH),
2
2
2
3
3
6.31 (1H, s, NH), 7.08 (1H, t, J = 8.2 Hz, CH quina), 7.22 (1H, d, J = 8.0 Hz, CH quina), 7.55
3
3
13
(
1H, t, J = 7.15 Hz, CH quina), 7.86 (1H, d, J = 7.8 Hz, CH quina), 10.10 (1H, s, NH); C
NMR (DMSOꢀd , 75 Hz): 15.8, 33.4, 36.8, 61.2, 101.1, 117.8, 122.0, 125.0, 126.3, 134.6, 150.9,
6
1
51.5, 162.4. C H N O , MW= 291.34 g/mol; t
= 1.73 min; MS (ESI+): m/z = 292
R,LCMS
15
21
3
3
[
M+H]+.
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General Procedure for synthesis of compounds 9 and 10. A solution of compound 7 (2.8 g,
.01 mol) or 8 (2.9 g, 0.01 mol) dissolved in sulfuric acid (40 mL) is stirred at room temperature
0
for 12h. The reaction mixture is poured in ice, alkalinized with 10N aqueous sodium hydroxide
solution (pH 10) and then acidified with diluted acetic acid solution to pH 5. The resulting
precipitate is filtered and recrystallized in ethanol.
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1H-imidazo[2,1-b]quinazolin-5-one (9). Yield 60%, 1.1 g. Offꢀwhite solid, mp 300ꢀ301 °C
2
6
1
4
3
(
Lit. 281ꢀ284 °C). H NMR (DMSOꢀd , 300 Hz): 7.14 (1H, td, J = 1.1 Hz and J = 6.9 Hz, CH
6
3
3
3
quina), 7.32 (1H, d, J = 2.25 Hz, CH), 7.42 (1H, d, J = 8.65 Hz, CH quina), 7.57 (1H, d, J =
4
3
4
2
.25 Hz, NCH), 7.63 (1H, td, J = 1.6 Hz and J = 8.65 Hz, CH quina), 8.11 (1H, dd, J = 1.6 Hz
3
13
and J = 8.25 Hz, CH quina), 9.32 (1H, s, NH). C NMR (DMSOꢀd , 75 Hz): 106.7, 112.6,
6
121.1, 122.4, 124.1, 127.1, 134.2, 146.0, 146.9, 157.7. C H N O, MW= 185.18 g/mol; t =
R,LCMS
10
7
3
1.43 min; MS (ESI+): m/z = 186 [M+H]+.
1,2-dihydro-1H-pyrimidino[2,1-b]quinazolin-6-one (10). Yield 89%, 1.75 g. Yellow solid, mp
1
4
>
400 °C. H NMR (DMSOꢀd , 300 Hz): 4.03 (2H, m, CH ), 5.58 (1H, m, CH), 7.07 (1H, td, J =
6 2
3
4
3
1
.1 Hz and J = 7.05 Hz, CH quina), 7.15 (1H, dd, J = 1.1 Hz and J = 8.25 Hz, CH quina), 7.32
4
3
4
3
(1H, dd, J = 2.05 Hz and J = 8.5 Hz, NCH), 7.54 (1H, td, J = 1.75 Hz and J = 8.15 Hz, CH
4
3
13
quina), 7.69 (1H, s, NH), 7.86 (1H, dd, J = 1.65 Hz and J = 7.85 Hz, CH quina). C NMR
(
DMSOꢀd , 75 Hz): 39.8, 110.0, 116.7, 119.3, 122.1, 124.3, 127.1, 135.1, 148.2, 150.3, 158.6.
6
C H N O, MW= 199.20 g/mol; t = 1.40 min; MS (ESI+): m/z = 200 [M+H]+.
R,LCMS
11
9
3
General Procedure for synthesis of compounds 12b-d. To a stirred solution of
methyltriphenylphosphonium iodide (20.7 g, 0.051 mol) in THF (100 mL) at 0 °C was added
potassium tertꢀbutoxide (8.5 g, 0.076 mol), and the mixture was stirred for another 1 h.
47
Commercial available (11bꢀc) or synthesized (11d) benzaldehyde (0.026 mol) in THF (25 mL)
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was added, and the reaction mixture was warmed to room temperature. After 24 h, saturated
aqueous ammonium chloride solution (150 ml) was added, the aqueous layer was extracted with
EtOAc (3 × 150 mL), and the combined organic phase was washed with brine, dried over
Na SO , and concentrated in vacuo. The resulting residue was purified by flash column
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chromatography on silica gel (DCM 100%).
-(3,4-dimethoxyphenyl)ethylene (12b). Yield 87%, 3.7 g. Yellow oil. H NMR (CDCl , 300
1
2
3
3
3
3
Hz): 3.90ꢀ3.92 (6H, 2s, OCH ), 5.16 (1H, dd, J = 0.8 Hz and J = 10.9 Hz, CH), 5.63 (1H, dd, J
3
2
3
2
= 0.8 Hz and J = 17.5 Hz, CH), 6.66 (1H, dd, J = 10.9 Hz and J = 17.5 Hz, CH), 6.82 (1H, d,
3
4
4
3
J = 8.0 Hz, ArH), 6.94 (1H, d, J = 1.85 Hz, ArH), 6.98 (1H, dd, J = 1.85 Hz and J = 8.0 Hz,
ArH). C H O ; MW= 164.20 g/mol.
10 12
2
1
2-(3,4,5-trimethoxyphenyl)ethylene (12c). Yield 90%, 4.5 g. Yellow oil. H NMR (CDCl , 300
3
3
3
3
Hz): 3.90 (9H, m, 9H, OCH ), 5.23 (1H, dd, J = 1.2 Hz and J = 11.2 Hz, CH), 5.67 (1H, dd, J
3
2
=
1.2 Hz and J = 18.0 Hz, CH), 6.65 (3H, m, CH, ArH). C H O ; MW= 194.22 g/mol.
11 14
3
2-(3-benzyloxy-4-methoxyphenyl)ethylene (12d). Yield 71%, 4.4 g. White solid, mp 67ꢀ69 °C
48
1
3
(
Lit. 68ꢀ69°C). H NMR (CDCl , 300 Hz): 3.85 (3H, s, OCH ), 5.08 (1H, dd, J = 0.85 Hz and
3 3
3
3
2
J = 10.8 Hz, CH), 5.25 (2H, s, OCH ), 5.51 (1H, d, J = 0.85 Hz and J = 11.75 Hz, CH), 6.58
2
3
2
3
4
(
1H, dd, J = 10.8 Hz and J = 11.75 Hz, CH), 6.82 (1H, d, J = 8.3 Hz, ArH), 6.92 (1H, dd, J =
3
4
1
.8 Hz and J = 8.45 Hz, ArH), 6.99 (1H, d, J = 1.8 Hz, ArH), 7.20ꢀ7.69 (5H, m, ArH benz).
C H O ; MW= 240.29 g/mol.
16
16
2
General Procedure for synthesis of compounds 13b-d. To a stirred solution of 12bꢀd (0.012
mol) in THF (30 mL) under a nitrogen atmosphere at 0 °C is added BH · THF (12 mL, 0.012
3
mol, 1.0 mol/L), and the mixture is stirred for 3h at room temperature. The reaction mixture is
cooled to 0 °C and water (1.7 mL) is added under magnetic stirring for 10 min before addition of
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0% aqueous sodium hydroxide solution (15 mL, 0.0375 mol) and 35% H O solution (15 mL).
2
2
The reaction mixture is stirred for another 30 min, quenched with saturated ammonium chloride
solution (100 mL) and then extracted with diethyl ether (3 × 50 mL). The combined organic
phase was washed with brine, dried over Na SO , and concentrated in vacuo. The resulting
0
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8
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8
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residue was purified by flash column chromatography on silica gel (DCM 100%).
1
2
-(3,4-dimethoxyphenyl)ethanol (13b). Yield 87%, 1.9 g. Clear oil. H NMR (CDCl , 300 Hz):
3
3
3
2.84 (2H, t, J = 6.5 Hz, CH ), 3.84 (2H, t, J = 6.6 Hz, CH ), 3.85 (3H, s, OCH ), 3.87 (3H, s,
2 2 3
OCH ), 6.75ꢀ6.83 (3H, m, ArH). C H O ; MW= 182.21 g/mol.
3
10 14
3
1
2
-(3,4,5-trimethoxyphenyl)ethanol (13c). Yield 77%, 1.95 g. Clear oil. H NMR (CDCl , 300
3
3
Hz): 2.81 (2H, t, J = 6.5 Hz, CH ), 3.81 (3H, s, OCH ), 3.85 (8H, m, CH , CH ), 6.44 (2H, s,
2
3
2
3
ArH). C H O ; MW= 212.24 g/mol.
1
1
16
4
2-(3-benzyloxy-4-methoxyphenyl)ethanol (13d). Yield 75%, 2.3 g. White solid, mp 42ꢀ44 °C
4
9
1
3
3
(
Lit. 68ꢀ69°C). H NMR (CDCl , 300 Hz): 2.76 (2H, t, J = 6.5 Hz, CH ), 3.78 (2H, t, J = 6.5
3 2
3
Hz, CH ), 3.88 (3H, s, OCH ), 5.16 (2H, s, OCH ), 6.80 (2H, m, ArH), 6.86 (1H, d, J = 8.8 Hz,
2
3
2
ArH), 7.30ꢀ7.50 (5H, m, ArH benz). C H O ; MW= 212.24 g/mol
1
1
16
4
General Procedure for synthesis of compounds 14a-d. A solution of methanesulfonyl
chloride (1 mL, 0.01 mol) in DCM (5 mL) is added dropwise to a solution of the commercial
available alcohol 13a or synthesized alcohol 13bꢀd (0.005 mol) and triethylamine (1.7 mL,
0.0125 mol) in DCM (15 mL) at 0 °C and stirred for 8 h at room temperature. The reaction
mixture is first washed with 1N aqueous hydrochloric acid solution (10 mL), then saturated
sodium bicarbonate solution (10 mL) and finally water (10 mL). The organic layer is dried,
filtered, concentrated in vacuo, and crystallized from cyclohexane.
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2
-(4-methoxyphenyl)ethylmethanesulfonate (14a). Yield 72%, 0.8 g. White solid, mp 35ꢀ36 °C
1
0
1
3
(
Lit. 35ꢀ36 °C). H NMR (CDCl , 300 Hz): 2.82 (3H, s, OSO CH ), 3.05 (2H, t, J = 6.5 Hz,
3
2
3
3
3
CH ), 3.80 (3H, s, OCH ), 4.38 (2H, t, J = 6.5 Hz, CH ), 6.90 (2H, d, J = 8.1 Hz, ArH), 7.15 (2H, d,
2
3
2
10
11
12
13
14
15
16
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46
47
48
49
50
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52
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55
56
57
58
59
60
3
J = 8.1 Hz, ArH). C H O S; MW= 230.28 g/mol.
1
0
14
4
2-(3,4-dimethoxyphenyl)ethylmethanesulfonate (14b). Yield 76%, 1.0 g. White solid, mp 42ꢀ
5
0
1
4
3°C (Lit. bp 168ꢀ169°C at 0.2 mm). H NMR (CDCl , 300 Hz): 2.89 (3H, s, OSO CH ), 3.01
3
2
3
3
3
(
2H, t, J = 6.5 Hz, CH ), 3.86 (3H, s, OCH ), 3.88 (3H, s, OCH ), 4.41 (2H, t, J = 6.5 Hz, CH ),
2 3 3 2
4
3
3
3
6
.76 (1H, dd, J = 1.7 Hz and J = 8.0 Hz, ArH), 6.80 (1H, d, J = 1.7 Hz, ArH), 6.84 (1H, d, J = 8.0
Hz, ArH). C H O S; MW= 260.30 g/mol.
1
1
16
5
2-(3,4,5-trimethoxyphenyl)ethylmethanesulfonate (14c). Yield 83%, 1.2 g. White solid, mp 76ꢀ
1
0
1
3
7
7 °C (Lit. 76ꢀ77 °C). H NMR (CDCl , 300 Hz): 3.02 (3H, s, OSO CH ), 3.10 (2H, t, J = 6.5
3
2
3
3
Hz), 3.85 (3H, s, OCH ), 3.88 (6H, s, OCH ), 4.43 (2H, t, J = 6.5 Hz, CH ), 6.46 (2H, s, ArH).
3
3
2
C H O S; MW= 290.33 g/mol.
12
18
6
2-(3-benzyloxy-4-methoxyphenyl)ethylmethanesulfonate (14d). Yield 69%, 1.2 g. White solid,
5
1
1
mp 95ꢀ97 °C (Lit. 106ꢀ107 °C). H NMR (CDCl , 300 Hz): 2.78 (3H, s, OSO CH ), 2.95 (2H, t,
3
2
3
3
3
J = 7.5 Hz, CH ), 3.89 (3H, s, OCH ), 4.34 (2H, t, J = 7.5 Hz, CH ), 5.17 (2H, s, OCH ), 6.75ꢀ6.90
2
3
2
2
(
3H, m, ArH), 7.30ꢀ7.50 (5H, m, ArH benz). C H O S; MW= 336.40 g/mol.
17 20 5
General Procedure for synthesis of compounds 15a-d and 16a-d. A solution of compound 9
or 10 (1 mmol) in anhydrous DMF (5 mL) is added dropwise at room temperature under nitrogen
to a suspension of sodium hydride (0.06 g, 1.5 mmol, 60% dispersion in mineral oil) in
anhydrous DMF (5 mL) and then stirred for 1 h at 60 °C. A solution of synthesized arylalkyl
methanesulfonate 14aꢀd (1.5 mmol) in anhydrous DMF (5 mL) is added dropwise to the
previous solution and stirred for 24 h at 60 °C. The reaction mixture is cooled in an ice bath and
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hydrolyzed with water (85 mL). The resulting precipitate is filtered and recrystallized in
EtOH/water (80/20).
N-[2-(4-methoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15a). Yield 23%, 74 mg. White
0
1
2
3
4
5
6
7
8
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solid, mp 133ꢀ135 °C. H NMR (CDCl , 300 Hz): 3.13 (2H, t, J = 7.4 Hz, CH ), 3.79 (3H, s,
3
2
3
3
3
OCH ), 4.34 (2H, t, J = 7.5 Hz, CH ), 6.57 (1H, d, J = 2.85 Hz, NCH), 6.84 (2H, d, J = 8.9 Hz,
3
2
3
3
3
ArH), 7.07 (2H, d, J = 8.9 Hz, ArH), 7.28 (1H, t, J = 7.6 Hz, CH quina), 7.45 (1H, d, J = 2.7 Hz,
3
13
CONCH), 7.65ꢀ7.72 (2H, m, CH quina), 8.37 (1H, d, J = 8.15 Hz, CH quina). C NMR (CDCl , 75
3
Hz): 34.4, 46.7, 55.2, 105.0, 114.2, 114.6, 119.2, 122.0, 125.5, 126.9, 129.5, 129.8, 134.0, 144.7,
1
50.0, 158.0, 158.6. C H N O MW= 319.35 g/mol; t
= 2.38 min; MS (ESI+): m/z = 320
R,LCMS
19
17
3
2;
[
M+H]+. HPLC purity 98.3%.
N-[2-(3,4-dimethoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15b). Yield 20%, 69 mg.
1
3
White solid, mp 160ꢀ162 °C. H NMR (CDCl , 300 Hz): 3.12 (2H, t, J = 7.2 Hz, CH ), 3.82
3
2
3
3
(
3H, s, OCH ), 3.85 (3H, s, OCH ), 4.36 (2H, t, J = 7.1 Hz, CH ), 6.59 (1H, d, J = 2.5 Hz,
3 3 2
3
3
NCH), 6.68 (2H, m, ArH), 6.78 (1H, d, J = 8.75 Hz, ArH), 7.28 (1H, t, J = 7.3 Hz, CH quina),
3
3
7
.45 (1H, d, J = 2.35 Hz, CONCH), 7.63ꢀ7.73 (2H, m, CH quina), 8.36 (1H, d, J = 8.0 Hz, CH
1
3
quina). C NMR (CDCl , 75 Hz): 34.8, 46.5, 55.9, 105.0, 111.3, 111.8, 114.6, 119.2, 120.9,
3
122.0, 125.5, 126.9, 129.9, 134.0, 144.7, 147.9, 149.1, 150.0, 157.9. C H N O MW= 349.38
20
19
3
3;
g/mol; tR,LCMS = 2.17 min; MS (ESI+): m/z = 350 [M+H]+. HPLC purity 99.4%.
N-[2-(3,4,5-trimethoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15c). Yield 22%, 83 mg.
1
3
Offꢀwhite solid, mp 172ꢀ174 °C. H NMR (CDCl , 300 Hz): 3.12 (2H, t, J = 7.2 Hz, CH ), 3.80
3
2
3
3
(
9H, s, OCH ), 4.39 (2H, t, J = 7.1 Hz, CH ), 6.38 (2H, s, ArH), 6.67 (1H, d, J = 2.6 Hz, NCH),
3
2
3
3
7.29 (1H, t, J = 7.5 Hz, CH quina), 7.50 (1H, d, J = 2.6 Hz, CONCH), 7.63ꢀ7.74 (2H, m, CH
3
13
quina), 8.36 (1H, d, J = 7.5 Hz, CH quina). C NMR (CDCl , 75 Hz): 35.7, 46.2, 56.1, 60.9,
3
105.2, 105.7, 114.6, 119.1, 122.1, 125.4, 127.0, 133.2, 134.1, 136.9, 144.8, 149.9, 153.4, 157.9.
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C H N O MW= 379.41 g/mol; t = 2.07 min; MS (ESI+): m/z = 380 [M+H]+. HPLC
R,LCMS
21
21
3
4;
purity 99.1%.
N-[2-(3-benzyloxy-4-methoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15d). Yield 26%,
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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33
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
2
38 mg. Offꢀwhite solid, mp 104ꢀ106 °C. H NMR (CDCl , 300 Hz): 3.07 (2H, t, J = 7.2 Hz,
3
3
3
CH ), 3.87 (3H, s, OCH ), 4.28 (2H, t, J = 7.2 Hz, CH ), 5.12 (2H, s, OCH ), 6.36 (1H, d, J =
2
3
2
2
4
3
4
2
.9 Hz, NCH), 6.65 (1H, dd, J = 1.8 Hz and J = 8.1 Hz, ArH), 6.71 (1H, d, J = 1.8 Hz, ArH),
3
6
.79 (1H, d, J = 8.12 Hz, ArH), 7.26ꢀ7.42 (7H, m, CONCH, CH quina, ArH benz), 7.65 (1H, d,
3
4
3
4
J = 7.4 Hz, CH quina), 7.71 (1H, td, J = 1.6 Hz and J = 6.8 Hz, CH quina), 8.37 (1H, dd, J =
3
13
1
.3 Hz and J = 8.4 Hz, CH quina); C NMR (CDCl , 75 Hz): 34.6, 46.4, 56.0, 71.0, 104.9,
3
1
12.0, 114.6, 114.8, 119.2, 121.6, 122.0, 125.5, 126.9, 127.2, 127.9, 128.5, 129.8, 134.0, 137.0,
144.7, 148.0, 148.7, 150.0, 157.9. C H N O MW= 425.48 g/mol; t = 2.69 min; MS
R,LCMS
26
23
3
3;
(ESI+): m/z = 426 [M+H]+.
N-[2-(4-methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a). Yield
1
3
2
3%, 77 mg. White solid, mp 114ꢀ116 °C. H NMR (CDCl , 300 Hz): 3.00 (2H, t, J = 7.6 Hz,
3
3
3
CH ), 3.80 (5H, m, CH , OCH ), 4.01 (2H, dd, J = 2.1 Hz and J = 3.5 Hz, NCH ), 5.34 (1H, td,
2
2
3
2
3
3
3
4
J = 3.5 Hz and J = 8.5 Hz, CH CH), 6.87 (2H, d, J = 8.6 Hz, ArH), 7.13 (1H, td, J = 1.05 Hz
2
3
3
3
and J = 7.15 Hz, CH quina), 7.22 (2H, d, J = 8.6 Hz, ArH), 7.36 (1H, d, J = 8.5 Hz, CONCH),
4
3
13
7
.49ꢀ7.59 (2H, m, CH quina), 8.07 (1H, dd, J = 1.2 Hz and J = 7.9 Hz, CH quina). C NMR
(CDCl , 75 Hz): 31.5, 46.9, 51.3, 55.3, 107.2, 114.0, 116.5, 120.2, 122.4, 125.2, 127.2, 129.8,
3
1
31.2, 134.6, 146.2, 149.5, 158.2, 159.5. C H N O , MW= 333.38 g/mol; t = 2.93 min;
2
0
19
3
2
R,LCMS
MS (ESI+): m/z = 334 [M+H]+. HPLC purity 97.9%.
N-[2-(3,4-dimethoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one
(16b).
1
3
Yield 20%, 72 mg. White solid, mp 144ꢀ146 °C. H NMR (CDCl , 300 Hz): 3.01 (2H, t, J = 7.6
3
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Hz, CH ), 3.80ꢀ3.90 (8H, m, CH , OCH ), 4.01 (2H, dd, J = 2.0 Hz and J = 3.3 Hz, NCH ),
2
2
3
2
3
3
3
5
.35 (1H, td, J = 3.5 Hz and J = 7.7 Hz, CH CH), 6.82 (3H, m, ArH), 7.14 (1H, t, J = 7.3 Hz,
2
3
CH quina), 7.33 (1H, d, J = 8.15 Hz, CH quina), 7.51ꢀ7.59 (2H, m, CONCH, CH quina), 8.09
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
13
(
1H, d, J = 7.95 Hz, CH quina). C NMR (CDCl , 75 Hz): 32.1, 46.9, 51.1, 55.9, 55.9, 107.1,
3
111.3, 112.0, 116.5, 120.3, 120.7, 122.5, 125.1, 127.3, 131.8, 134.6, 146.2, 147.6, 149.0, 149.4,
59.5 . C H N O , MW= 363.41 g/mol; t = 2.65 min; MS (ESI+): m/z = 364 [M+H]+.
1
21
21
3
3
R,LCMS
HPLC purity 97.4%.
N-[2-(3,4,5-trimethoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one
(16c).
1
3
Yield 22%, 86 mg. White solid, mp 156ꢀ158 °C. H NMR (CDCl , 300 Hz): 3.00 (2H, t, J = 7.4
3
3
3
Hz, CH ), 3.84 (5H, m, CH , OCH ), 3.88 (6H, s, OCH ), 4.04 (2H, dd, J = 1.95 Hz and J =
2
2
3
3
3
3
3.25 Hz, NCH ), 5.37 (1H, td, J = 3.55 Hz and J = 8.3 Hz, CH CH), 6.53 (2H, s, ArH), 7.14
2
2
4
3
3
(
(
(
1H, td, J = 1.0 Hz and J = 8.1 Hz, CH quina), 7.32 (1H, d, J = 8.1 Hz, CH quina), 7.51ꢀ7.60
4
3
13
2H, m, CONCH, CH quina), 8.08 (1H, dd, J = 1.15 Hz and J = 7.8 Hz, CH quina). C NMR
CDCl , 75 Hz): 32.9, 46.9, 50.9, 56.1, 60.9, 105.6, 107.1, 116.5, 120.3, 122.5, 125.0, 127.3,
3
1
34.7, 135.0, 136.5, 146.2, 149.4, 153.3, 159.5. C H N O , MW= 393.43 g/mol; t
= 2.67
R,LCMS
2
2
23
3
4
min; MS (ESI+): m/z = 394 [M+H]+. HPLC purity 97.7%.
N-[2-(3-benzyloxy-4-methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one
1
(
16d). Yield 26%, 114 mg. White solid, mp 139ꢀ141 °C. H NMR (CDCl , 300 Hz): 2.94 (2H, t,
3
3
3
J = 7.6 Hz, CH ), 3.74 (2H, t, J = 7.15 Hz, CH ), 3.88 (5H, m, NCH , OCH ), 5.16 (2H, s,
2
2
2
3
3
3
3
OCH ), 5.29 (1H, td, J = 3.5 Hz and J = 7.15 Hz, CH CH), 6.84 (3H, m, ArH), 7.13 (1H, t, J =
2
2
7
.5 Hz, CH quina), 7.29ꢀ7.38 (4H, m, CH quina, ArH benz), 7.42ꢀ7.50 (3H, m, CONCH, ArH
4
3
4
3
benz), 7.56 (1H, td, J = 1.45 Hz and J = 8.5 Hz, CH quina), 8.08 (1H, dd, J = 1.25 Hz and J =
13
7
.9 Hz, CH quina); C NMR (CDCl , 75 Hz): 20.3, 32.7, 40.4, 46.3, 51.4, 55.9, 70.4, 113.3,
3
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14.4, 116.2, 121.1, 121.6, 124.8, 126.6, 128.2, 128.8, 132.8, 134.4, 137.7, 146.6, 148.9, 149.5,
149.8, 161.9. C H N O , MW= 439.50 g/mol; t = 2.95 min; MS (ESI+): m/z = 440
R,LCMS
27 25
3
3
[
M+H]+.
General Procedure for synthesis of compounds 15e and 16e. A solution of 15d or 16d (0.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
32
33
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37
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43
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54
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56
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58
59
60
mmol) in a 33% solution of hydrobromic acid (3 mL) and glacial acetic acid (5 mL) is stirred at
room temperature until starting material disappeared (followed by TLC). After cooling, the
reaction mixture was diluted with water (50 mL) and the resulting precipitate was extracted with
EtOAc (3 x 20 mL). The organic layers were combined, washed with an aqueous 10% NaHCO3
solution, dried (MgSO ), and evaporated to give a white solid purified by recrystallization from
4
ethanol.
N-[2-(3-hydroxy-4-methoxy-phenyl)ethyl]imidazo[2,1-b]quinazoline-5-one (15e). Yield 63%,
1
3
1
05 mg. White solid, mp 133ꢀ135 °C. H NMR (CDCl , 300 Hz): 3.07 (2H, t, J = 6.9 Hz, CH ),
3 2
3
4
3
3
.84 (3H, s, OCH ), 4.32 (2H, t, J = 6.95 Hz, CH ), 6.55 (1H, dd, J = 2.05 Hz and J = 8.2 Hz,
3
2
4
3
3
ArH), 6.59 (1H, d, J = 2.7 Hz, ArH), 6.73 (1H, d, J = 8.2 Hz, ArH), 6.79 (1H, d, J = 2.05 Hz,
3
NCH), 7.27 (1H, m, CH quina), 7.44 (1H, d, J = 2.75 Hz, CONCH), 7.63ꢀ7.72 (2H, m, CH
4
3
13
quina), 8.34 (1H, dd, J = 1.15 Hz and J = 8.4 Hz, CH quina). C NMR (CDCl , 75 Hz): 34.6,
3
4
6.5, 55.9, 105.0, 110.9, 114.6, 114.9, 119.2, 120.3, 121.9, 125.6, 126.9, 130.6, 133.9, 144.7,
45.7, 145.9, 150.0, 157.9. C H N O , MW= 335.35 g/mol; t = 2.01 min; MS (ESI+):
1
19
17
3
3
R,LCMS
m/z = 336 [M+H]+. HPLC purity 98.6%.
N-[2-(3-hydroxy,4-methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one
1
3
(
16e). Yield 56%, 98 mg. White solid, mp 140ꢀ142 °C. H NMR (CDCl , 300 Hz): 2.96 (2H, t, J
3
3
3
3
= 7.7 Hz, CH ), 3.79 (2H, t, J = 7.7 Hz, CH ), 3.89 (3H, s, OCH ), 4.03 (2H, dd, J = 2.1 Hz and J
2
2
3
3
3
=
3.4 Hz, NCH ), 5.35 (1H, td, J = 3.4 Hz and J = 8.4 Hz, CH CH), 5.63 (1H, s, OH), 6.78 (2H, m,
2 2
3
4
3
ArH), 6.91 (1H, d, J = 1.8 Hz, ArH), 7.12 (1H, td, J = 1.2 Hz and J = 8.15 Hz, CH quina), 7.36
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5
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6
4
3
(
1H, dd, J = 1.2 Hz and J = 8.2 Hz, CH quina), 7.49ꢀ7.59 (2H, m, CONCH, CH quina), 8.07 (1H,
4
3
13
dd, J = 1.6 Hz and J = 8.25 Hz, CH quina); C NMR (CDCl , 75 Hz): 20.3, 32.6, 40.4, 46.3, 51.6,
3
55.9, 113.3, 115.8, 116.1, 121.4, 121.6, 124.8, 126.6, 130.6, 134.4, 145.3, 147.9, 148.9, 149.8, 161.9.
0
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7
8
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0
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7
8
9
0
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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
C H N O , MW= 349.38 g/mol; t = 2.17 min; MS (ESI+): m/z = 350 [M+H]+. HPLC
R,LCMS
20
19
3
3
purity 98.5%.
General Procedure for synthesis of compounds 17a-d and 18a-d. A solution of 15aꢀd or 16aꢀ
d (0.5 mmol) in EtOH (100 mL) is stirred with 10% Pd/C catalyst and ammonium formate (15
mmol) at 60°C until starting material disappeared (followed by TLC). The warm solution was
filtered on a pad of celite and the filtrate was evaporated. The residue was recrystallized from
methanol.
N-[2-(4-methoxyphenyl)ethyl]-2,3-dihydro-imidazo-[2,1-b]quinazolin-5-one (17a). Yield 65%,
1
3
1
04 mg. White solid, mp 98ꢀ100 °C. H NMR (DMSOꢀd , 300 Hz): 2.85 (2H, t, J = 7.45 Hz,
6
3
3
CH ), 3.60 (4H, m, NCH ), 3.71 (3H, s, OCH ), 4.02 (2H, t, J = 7.9 Hz, CH ), 6.87 (2H, d, J = 8.9
2
2
3
2
3
3
3
Hz, ArH), 7.12 (1H, t, J = 7.45, CH quina), 7.22 (2H, d, J = 8.9 Hz, ArH), 7.29 (1H, d, J = 7.9 Hz,
3
3
13
CH quina), 7.57 (1H, t, J = 8.4 Hz, CH quina), 7.92 (1H, d, J = 7.95 Hz, CH quina). C NMR
(
DMSOꢀd , 75 Hz): 32.2, 44.8, 46.1, 55.4, 114.3, 117.8, 122.3, 125.0, 126.2, 130.1, 131.2, 134.4,
6
151.3, 153.0, 158.2, 160.6. C H N O , MW= 321.37 g/mol; t = 2.21 min; MS (ESI+):
R,LCMS
19
19
3
2
m/z = 322 [M+H]+. HPLC purity 99.0%.
N-[2-(3,4-dimethoxyphenyl)ethyl]-2,3-dihydro-imidazo-[2,1-b]quinazolin-5-one (17b). Yield
1
3
53%, 93 mg. White solid, mp 84ꢀ86 °C. H NMR (CDCl , 300 Hz): 2.95 (2H, t, J = 7.45 Hz,
3
3
3
CH ), 3.53 (2H, t, J = 8.6 Hz, NCH ), 3.75 (2H, t, J = 6.85 Hz, NCH ), 3.86 (3H, s, OCH ), 3.87
2
2
2
3
3
4
3
(
3H, s, OCH ), 4.10 (2H, t, J = 8.05 Hz, CH ), 6.81 (3H, m, ArH), 7.16 (1H, td, J = 1.15 Hz and J
3
2
4
3
4
=
8.05 Hz, CH quina), 7.39 (1H, dd, J = 0.65 Hz and J = 8.2 Hz, CH quina), 7.57 (1H, td, J = 1.6
3
4
3
13
Hz and J = 8.45 Hz, CH quina), 8.11 (1H, dd, J = 1.25 Hz and J = 7.9 Hz, CH quina). C NMR
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(
CDCl , 75 Hz): 33.2, 40.4, 45.1, 46.0, 55.9, 111.3, 111.8, 117.7, 120.7, 122.5, 124.8, 126.5,
3
1
31.1, 134.3, 147.7, 149.1, 150.9, 152.4, 161.3. C H N O , MW= 351.40 g/mol; t
= 2.05
R,LCMS
20 21
3
3
min (98.9%); MS (ESI+): m/z = 352 [M+H]+. HPLC purity 97.9%.
N-[2-(3,4,5-trimethoxyphenyl)ethyl]-2,3-dihydro-imidazo-[2,1-b]quinazolin-5-one (17c). Yield
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
3
52%, 93 mg. White solid, mp 144ꢀ146 °C. H NMR (CDCl , 300 Hz): 2.96 (2H, t, J = 7.4 Hz,
3
3
3
CH ), 3.59 (2H, t, J = 8.2 Hz, NCH ), 3.78 (2H, t, J = 7.4 Hz, NCH ), 3.82 (3H, s, OCH ), 3.85
2
2
2
3
3
3
(
6H, s, OCH ), 4.14 (2H, t, J = 8.2 Hz, CH ), 6.52 (2H, s, ArH), 7.18 (1H, t, J = 7.75 Hz, CH
3
2
3
3
3
quina), 7.40 (1H, d, J = 8.1 Hz, CH quina), 7.58 (1H, t, J = 7.1 Hz, CH quina), 8.13 (1H, d, J =
1
3
7.75 Hz, CH quina). C NMR (CDCl , 75 Hz): 33.9, 40.4, 45.0, 45.7, 56.2, 60.9, 105.5, 117.7,
3
1
22.6, 124.8, 126.5, 134.2, 134.3, 150.9, 152.4, 153.3, 161.3. C H N O , MW= 381.42 g/mol;
21 23 3 4
tR,LCMS = 2.05 min (95.6%); MS (ESI+): m/z = 382 [M+H]+. HPLC purity 95.9%.
N-[2-(3-hydroxy-4-méthoxy-phényl)éthyl]-2,3-dihydro-imidazo[2,1-b]quinazoline-5-one (17d).
1
3
Yield 45%, 151 mg. White solid, mp 210ꢀ212 °C. H NMR (CDCl , 300 Hz): 2.92 (2H, t, J =
3
3
3
7.05 Hz, CH ), 3.53 (2H, t, J = 8.5 Hz, NCH ), 3.72 (2H, t, J = 7.05 Hz, NCH ), 3.87 (3H, s,
2
2
2
3
4
3
OCH ), 4.11 (2H, t, J = 7.5 Hz, CH ), 5.77 (1H, s, OH), 6.74 (1H, dd, J = 2.05 Hz and J = 8.2
3
2
3
4
4
Hz, ArH), 6.80 (1H, d, J = 8.2 Hz, ArH), 6.88 (1H, d, J = 2.05 Hz, ArH), 7.17 (1H, td, J = 1.15
3
4
3
Hz and J = 7.15 Hz, CH quina), 7.41 (1H, dd, J = 0.6 Hz and J = 8.3 Hz, CH quina), 7.58 (1H, td,
4
3
4
3
13
J = 1.5 Hz and J = 7.1 Hz, CH quina), 8.12 (1H, dd, J = 1.15 Hz and J = 7.95 Hz, CH quina). C
NMR (CDCl , 75 Hz): 33.1, 40.4, 45.2, 46.2, 55.9, 110.8, 114.9, 117.8, 120.1, 122.4, 124.9,
3
126.4, 131.9, 134.2, 145.4, 145.7, 151.0, 152.4, 161.3. C H N O MW= 337.37 g/mol; t
15
19
3
3;
R,LCMS
=
1.87 min (96.6%); MS (ESI+): m/z = 338 [M+H]+. HPLC purity 98.4%.
N-[2-(4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one
(18a).
1
Yield 69%, 115 mg. White solid, mp 242ꢀ244 °C. H NMR (CDCl , 300 Hz): 1.97 (2H, m, CH ),
3
2
3
3
3
2
.99 (2H, t, J = 7.5 Hz, NCH2), 3.26 (2H, t, J = 6.9 Hz, NCH2), 3.80 (3H, s, OCH ), 3.86 (2H, t, J
3
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4
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5
5
5
5
5
5
5
5
5
5
6
2
3
4
=
7.1 Hz, CH2), 4.05 (2H, t, J = 6.8 Hz, CH2), 6.85 (2H, d, J = 8.65 Hz, ArH), 7.11 (1H, td, J = 1.3
3
3
4
3
Hz and J = 7.1 Hz, CH quina), 7.18 (2H, d, J = 8.4 Hz, ArH), 7.36 (1H, dd, J = 0.5 Hz and J = 8.3
4
3
4
Hz, CH quina), 7.57 (1H, td, J = 1.6 Hz and J = 7.0 Hz, CH quina), 8.09 (1H, dd, J = 1.15 Hz and
0
1
2
3
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1
2
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1
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7
8
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1
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
13
J = 8.0 Hz, CH quina). C NMR (CDCl , 75 Hz): 20.7, 32.5, 40.0, 47.0, 52.2, 55.3, 113.9, 116.3,
3
1
21.7, 124.7, 126.8, 129.9, 131.4, 134.1, 148.3, 149.6, 158.2, 162.7. C H N O MW= 335.39
20 21 3 2;
g/mol; tR,LCMS = 2.59 min (96.7%); MS (ESI+): m/z = 336 [M+H]+. HPLC purity 99.1%.
N-[2-(3,4-dimethoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one
1
(
18b). Yield 52%, 95 mg. White solid, mp 132ꢀ134 °C. H NMR (CDCl , 300 Hz): 1.98 (2H, m,
3
3
3
CH2), 2.99 (2H, t, J = 7.5 Hz, NCH ), 3.26 (2H, t, J = 5.8 Hz, NCH ), 3.87ꢀ3.91 (8H, m, CH ,
2
2
2
2
4
3
OCH ), 4.04 (2H, t, J = 5.85 Hz, CH ), 6.68 (3H, m, ArH), 7.11 (1H, td, J = 1.1 Hz and J = 8.0
3
2
3
4
3
Hz, CH quina), 7.34 (1H, d, J = 8.15 Hz, CH quina), 7.56 (1H, td, J = 1.6 Hz and J = 8.3 Hz,
4
3
13
CH quina), 8.09 (1H, dd, J = 1.5 Hz and J = 8.0 Hz, CH quina). C NMR (CDCl , 75 Hz): 20.7,
3
3
3.0, 40.0, 46.9, 51.9, 55.8, 55.9, 111.2, 112.1, 116.3, 120.9, 121.7, 124.6, 126.8, 132.0, 134.1,
147.6, 148.3, 148.9, 149.6, 162.6. C H N O MW= 365.42 g/mol; t = 2.38 min (98.2%);
2
1
23
3
3;
R,LCMS
MS (ESI+): m/z = 366 [M+H]+. HPLC purity 98.5%.
N-[2-(3,4,5-trimethoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one
1
(18c). Yield 43%, 85 mg. White solid, mp 136ꢀ138 °C. H NMR (CDCl , 300 Hz): 2.01 (2H, m,
3
3
3
CH2), 2.99 (2H, t, J = 7.45 Hz, NCH ), 3.31 (2H, t, J = 5.8 Hz, NCH ), 3.82 (3H, s, OCH ), 3.84
2
2
3
3
3
(
6H, s, OCH ), 3.91 (2H, t, J = 7.0 Hz, CH ), 4.06 (2H, t, J = 5.95 Hz, CH ), 6.49 (2H, s, ArH),
3 2 2
4
3
3
7.11 (1H, td, J = 1.15 Hz and J = 8.0 Hz, CH quina), 7.33 (1H, d, J = 8.2 Hz, CH quina), 7.56
4
3
4
3
(
1H, td, J = 1.7 Hz and J = 8.45 Hz, CH quina), 8.09 (1H, dd, J = 1.25 Hz and J = 8.0 Hz, CH
13
quina). C NMR (CDCl , 75 Hz): 20.7, 33.8, 40.1, 46.8, 51.7, 56.1, 60.9, 105.7, 116.3, 121.8,
3
1
24.6, 126.9, 134.1, 135.2, 136.5, 148.3, 149.5, 153.3, 162.6. C H N O ; MW= 395.45 g/mol;
22 25 3 4
tR,LCMS = 2.38 min (97.3%); MS (ESI+): m/z = 396 [M+H]+. HPLC purity 98.1%.
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N-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydropyrimidino[2,1-b]quinazolin-6-one
1
(
18d). Yield 51%, 89 mg. White solid, mp 153ꢀ155 °C. H NMR (DMSOꢀd , 300 Hz): 1.93 (2H,
6
3
3
m, CH ), 2.81 (2H, t, J = 7.7 Hz, NCH ), 3.31 (2H, t, J = 5.3 Hz, NCH ), 3.73 (5H, m, CH ,
2
2
2
2
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
3
4
3
OCH ), 3.93 (2H, t, J = 5.8 Hz, CH ), 6.65 (1H, dd, J = 1.9 Hz and J = 8.1 Hz, ArH), 6.73 (1H,
3
2
4
3
4
3
d, J = 1.9 Hz, ArH), 6.84 (1H, d, J = 8.3 Hz, ArH), 7.08 (1H, t, J = 1.1 Hz and J = 8.1 Hz, CH
4
3
4
3
quina), 7.27 (1H, dd, J = 1.1 Hz and J = 7.75 Hz, CH quina), 7.57 (1H, t, J = 1.6 Hz and J = 8.4
4
3
13
Hz, CH quina), 7.89 (1H, dd, J = 1.15 Hz and J = 7.95 Hz, CH quina), 8.90 (1H, s, OH); C
NMR (CDCl , 75 Hz): 20.3, 32.3, 40.3, 46.4, 51.8, 56.1, 112.8, 116.2, 116.5, 119.7, 121.6,
3
1
24.9, 126.6, 132.4, 134.4, 146.5, 146.9, 148.9, 149.8, 161.9. C H N O ; MW= 351.39 g/mol;
20 21 3 3
tR,LCMS = 2.10 min (99.2%); MS (ESI+): m/z = 352 [M+H]+. HPLC purity 96.6%.
General Procedure for synthesis of compounds 22 and 23. To a mixture of compound 20 or
2
1 (0.02 mol) and triethylamine (5.6 mL, 0.04 mol) in DCM (200 mL) is added under ice cooling
and dropwise 2ꢀnitrobenzenesulfonyl chloride 19 (3.5 g, 0.016 mol) dissolved in DCM (50 mL)
and the mixture is allowed to stir at room temperature for 2h. The reaction mixture is hydrolyzed,
extracted with DCM (2 x 50 mL) and washed with 10% aqueous sodium thiosulfate solution.
The organic layers were combined, dried (MgSO ), and evaporated to give a colorless solid
4
purified by recrystallization from methanol.
N-(2-nitrophenylsulfonyl)-2-methylsulfanyl-4,5-dihydro-imidazole (22). Yield 62%, 3.0 g.
5
2
1
Colorless solid, mp 184ꢀ185 °C (Lit. 185ꢀ186 °C). H NMR (CDCl , 300 Hz): 2.47 (3H, s,
3
3
3
SCH ), 3.91 (2H, t, J = 7.5 Hz, NCH ), 4.10 (2H, t, J = 7.5 Hz, NCH ), 7.68ꢀ7.82 (3H, m,
3
2
2
4
3
ArH), 8.28 (1H, dd, J = 2.4 Hz and J = 7.3 Hz, ArH). C H N O S ; MW= 301.34 g/mol.
10 11
3
4 2
N-(2-nitrophenylsulfonyl)-2-benzylsulfanyl-1,4,5,6-tetrahydropyrimidine (23). Yield 91%, 5.7
27
1
g. Colorless solid, mp 68ꢀ70 °C (Lit. 85 °C). H NMR (CDCl , 300 Hz): 1.99 (2H, m, CH ),
3
2
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