Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal

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Specific inhibitors of HIV capsid assembly binding to the C-terminal domain of the capsid protein: evaluation of 2-arylquinazolines as potential antiviral compounds Ales Machara, Vanda Lux, Milan Kozisek, Klara Grantz-Saskova, Ondrej Stepanek, Martin Kotora, Kamil Parkan, Marcela Pavova, Baerbel Glass, Peter Sehr, Joe Lewis, Barbara Muller, Hans-Georg Kraeusslich, and Jan Konvalinka J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01089 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 21, 2015

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Journal of Medicinal Chemistry

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Specific inhibitors of HIV capsid assembly binding to the C-terminal domain of the capsid protein: evaluation of 2-arylquinazolines as potential antiviral compounds

Aleš Machara,1#, Vanda Lux,2,#$, Milan Kožíšek3, Klára Grantz Šašková,3,4, Ondřej Štěpánek1, Martin Kotora1, Kamil Parkán3, Marcela Pávová2,3, Bärbel Glass2, Peter Sehr5, Joe Lewis5, Barbara Müller2, Hans-Georg Kräusslich2*, Jan Konvalinka,3,4* 1

Department of Organic Chemistry, Faculty of Science, Charles University, Prague, Czech

Republic; 2Department of Infectious Diseases, Virology, University Heidelberg, Germany; 3

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech

Republic, Gilead Sciences and IOCB Research Center, Prague, Czech Republic; 4Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic; 5Chemical Biology Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany

#

these authors contributed equally to this work

$

current address:

Faculty of Biology, Centre for Medical Biotechnology, University Duisburg–Essen, 45117 Essen, Germany

*To whom correspondence should be addressed: [email protected], [email protected]

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Abstract: Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy, which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors, based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70,000 compounds from different libraries identified 2arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA, to compete with the known peptide inhibitor, and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA, and were found to block viral replication at low micromolar concentrations.



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Introduction: Human Immunodeficiency Virus type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). The introduction of antiviral combination therapy targeting different viral enzymes into clinical practice has led to a dramatic decrease of mortality and prolonged life expectancy of HIV-positive patients. Nevertheless, the clinical benefit of these drugs can be compromised by the emergence of drug-resistant viral strains.1 Therefore, there is still a need to search for new anti-HIV-1 therapeutic drugs, less prone to resistance development and possessing high antiviral potential that would enlarge our anti-HIV armament. A cell infected with HIV-1 is programmed to produce all structural components of the virus and assemble them into progeny virus particles, which are then released to infect other cells. The viral Gag polyprotein alone is necessary and sufficient to form virus like particles. HIV-1 virions comprise approximately 2,500 Gag molecules organized into a semi-spherical protein shell.2 These polyproteins radially line the inside of the viral membrane and form the immature core. Gag multimerization is mainly driven by intermolecular interactions between the CA (capsid), spacer peptide 1 (SP1) and NC (nucleocapsid) region of neighboring Gag molecules.3, 4, 5 During or shortly after release of the immature particle, Gag is processed by the viral protease (PR) into MA (matrix), CA, SP1, NC, spacer peptide 2 (SP2) and a protein of 6 kDa (p6). Processed CA comprises two independently folded domains, the N-terminal CA NTD domain (residues 1−146) and the C-terminal CA CTD domain (residues 151−231) that are connected by a short flexible linker. Hexamers and pentamers of CA form the fullerene-type conical core of the mature virion which encloses the viral genome.2 The morphological transformation from immature to mature architecture is essential for viral infectivity. The HIV PR therefore constitutes an excellent target for antiretroviral therapy with nine PR inhibitors currently in clinical use.1, 6 The CA domain of Gag thus forms the main protein interfaces in both the immature and the mature core, but using almost completely different contact surfaces. The currently favored model suggests that maturation occurs by disassembly of the immature lattice and reassembly of the mature lattice. Accordingly, HIV-1 particle formation involves two consequent assembly steps, both of which represent potential targets for anti-HIV intervention. 3 ACS Paragon Plus Environment

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Currently, there are no assembly inhibitors in clinical use, but several low molecular weight CA-binding compounds have been identified. All of these compounds bind to the HIV-1 CA NTD, and some affect CA assembly as well.7 Pioneering work by Summers and colleagues identified CAP-1, a small molecule that weakly binds into a hydrophobic pocket normally occupied by Phe32 of the neighboring CA molecule in the mature lattice. CAP-1 binding thereby distorts the loop between helices 3 and 4 of CA NTD (Figure 1).8 Other compounds binding to the same pocket include acylhydrazones and thioureas.9,10,11 These compounds also bind with micromolar affinity and display weak antiviral activity. High-throughput assays helped to identify two structurally diverse groups of assembly inhibitors, benzodiazepines and benzimidazoles12,13,14, which bind to CA NTD and induce the formation of a pocket that overlaps with the CAP-1 binding site (Figure 1). Some compounds from these groups exhibit moderate to potent anti- HIV-1 activities in tissue culture (EC50 < 100 nM).13 More recently, other compounds binding near the flexible cyclophilin A-binding loop in CA NTD (Figure 1) were identified. 15

Figure 1: Illustrative representation of the HIV-1 capsid structure (PDB 2LF4)16 with highlighted ligand binding sites.



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A different region in CA NTD is targeted by PF-3450074 (PF74), which binds hexameric CA with an affinity of 120 nM and inhibits HIV-1 replication in tissue culture at low nanomolar concentrations.7, 17, 18 Its binding pocket is formed by helices 3, 4, 5 and 7 (Figure 1), and PF74 binding does not cause a conformational change in the structure of CA NTD. This compound has been suggested to inhibit HIV replication in the early phase of infection by destabilizing the capsid structure and triggering premature uncoating of the viral core17, while recent experiments indicated that its main mode of action may be interference with nuclear targeting of the incoming capsid.18-19 All described compounds bind to CA NTD, and no small molecule inhibitors targeting CA CTD have been identified so far. Sticht et al. reported a 12mer peptide, termed CAI (capsid assembly inhibitor), that binds to CA CTD (Figure 1) and inhibits assembly of immature- and mature-like particles in vitro.20 X-ray structure analysis of the CA CTD-CAI complex showed that the peptide inserts itself as an amphipathic helix into a conserved hydrophobic groove in CA CTD, thereby altering the dimer interface.21 CAI thus points to a unique reactive groove in CA and may serve as a lead compound for HIV assembly inhibitors (Figure 2A). Mutating CA residues contacting CAI in the complex (Y169 or L211) yielded reduced affinity of the resulting CA proteins for the peptide, and rendered the variant Gag proteins unable to assemble mature particles in vitro or in tissue culture. These mutations apparently locked CA in the immature conformation and thereby prevented maturation to the infectious virion.22 X-ray crystallographic analyses of the variant CA CTD domains revealed that these alterations induced the same allosteric changes at the CA dimer interface that were observed in the CA CTD/CAI complex.24 Due to its low cell permeability, CAI could not be directly used to inhibit HIV-1 replication in cell culture, but a CAI derivative converted into a cell-penetrating peptide by hydrocarbon stapling (NYAD-1) displayed some anti-HIV-1 activity in cell culture.23 This approach validated the CAI-binding pocket as target for HIV-1 assembly inhibitors, but the resulting compound still suffers from the drawbacks of peptidic inhibitors including poor general and lack of oral bioavailability, as well as short half-life. Accordingly, we screened for small molecules targeting the same pocket as CAI as lead compounds for assembly inhibition. Screening >70,000 compounds in a newly developed high-throughput competition assay yielded two hits, which were subsequently modified to determine CA binding and antiviral potency (see Supplementary Figure 1). Here, we report the identification and characterization of several compounds binding to CA CTD and inhibiting HIV-1 replication in tissue culture at moderate potency.


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Figure 2: (A) Structure of the HIV-1 CA hexamer in the mature lattice (PDB 3MGE)24, one CA monomer is highlighted (blue, NTD; green, CTD). The second colored CA represents a monomer from the neighboring hexamer. The inset shows the CAI peptide (in red) bound to CA CTD. (B) Principle of the AlphaScreen high-throughput assay based on CAI binding to CA CTD.

Results and Discussion :

Development and validation of binding assay and compound screening The new assay for screening of compound libraries for HIV-1 assembly inhibitors is based on the amplified luminescent proximity assay system (AlphaScreen), where interaction of CAI on donor beads with the CTD of CA on acceptor beads brings the two beads into close proximity, thus creating a robust signal. For this, biotinylated CAI was non-covalently bound to donor beads coated with streptavidin. CA was expressed as a glutathione S-transferase (GST) fusion protein in E.coli, purified and bound to acceptor beads coated with glutathione. 6 ACS Paragon Plus Environment


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Upon excitation of the donor, ambient oxygen is converted to singlet oxygen which diffuses approximately 200 nm within its half-life. If an acceptor bead is present within that distance, energy is transferred from singlet oxygen to the acceptor and an emission signal can be detected (Figure 2). Accordingly, interaction of CAI and CA resulted in luminescence due to close proximity of donor and acceptor beads. This signal could be competed by soluble CAI, but not by a scrambled control peptide with the same amino acid composition as CAI (data not shown). These results validated the newly developed assay for screening of small molecule competitors of the CAI-CA CTD interaction as potential assembly inhibitors. To validate the assay for high-throughput screening, a pilot screen in two independent replication was undertaken with a subset of 3,200 and 3,000 compounds from DiverSet Chembridge library and LeadQuest Tripos Library, respectively. The validation data are shown only for the Chembridge library, as it contains the compound 16, described in this work and as the results for Tripos Library were of similar quality. Inhibition values for each of the 3,200 compounds in either replicate are compared in a scattered plot (Supplementary Figure 1B). The correlation of results obtained in both runs with a slope of 1.021 and an R2 value for linear regression of 0.8 indicates good reproducibility of results. The assay showed a high signal-to-noise ratio (150,000 versus 200 = 750) and low variation within the controls, CV values were in the range 3.3 – 4.1%. From the controls in columns 1, 2, 23, and 24, the Z′ factor as a parameter for the quality of the assay was calculated for each plate (Supplementary Figure 1C). The Z’ values were in the range of 0.88 to 0.92, which proves excellent assay performance and its suitability for highthroughput screening. A total of >70,000 chemically diverse compounds from two small molecule libraries (DiverSet Chembridge library, 20,000 compounds; LeadQuest Tripos Library 50,000 compounds), were screened at a concentration of 40 µM in vitro using this assay. Assay reproducibility was determined for the pilot screen (see Supplementary information, Supplementary Figure 1). In summary, 151 and 184 hits from DiverSet Chembridge library and LeadQuest Tripos Library, respectively, displayed inhibition of >40% in the initial screen. These compounds were re-orderd and analysed in titration experiments from 0.2 - 200 µM for solubility using nephelometry as well as for their half maximal inhibitory concentration (IC50 value) in the AlphaScreen. Compounds were further subjected to a counterscreen with biotinylated glutathione S-transferase mixed with donor and acceptor beads to identify non-specific compounds that affected the AlphaScreen signal due to reasons other than interference with CA-CAI binding. Inhibitory activity was reproduced for ~ 50% of 7 ACS Paragon Plus Environment

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of initial hits. From these, 94,4% of DiverSet Chembridge hits and all LeadQuest Tripos compounds were rejected based on the results from the counterscreen. The remaining hits and all chemical derivatives thereof contained in the original Chembridge library were compared for inhibition of CA-CAI binding. A set of five compounds carrying an arylquinazoline moiety showed IC50 values in the micromolar range. The most potent hit of this group, compound 16 (Figure 3), served as the basis for subsequent optimization by medicinal chemistry.

Figure 3: Lead compound identified by the library screening using the AlphaScreening test, 2-arylquinazoline 16.

Compound synthesis The derivatives of the lead compound 16 were synthesized efficiently following the procedures shown in Figure 4. Anthranilamide was acylated with selected acyl chloridepyridine compounds to form diamides 1, 2, 7 which were cyclized upon heating with aqueous potassium hydroxide. Treatment of the quinazolinones 3, 4, 8 with phosphoryl chloride in dimethylformamide (DMF) at 80 °C yielded 4-chloroquinazolines 5, 6, 9. These key intermediates were used as substrates for further modifications, which led to a diverse series of desired compounds. Nucleophilic displacement of chloro atom at C-4 in 5, 6, 9 with selected anilines in 1,4-dioxane was performed to obtain 4-N-arylquinazolines 10-36. Anilines were either purchased or prepared from appropriate nitrobenzenes (see Supporting Information). Reactions were performed with or without the presence of hydrogen chloride, depending on nucleophilicity of the used anilines. Conversion of ethyl ester 17 to amide 21 was carried out by heating with excess of aminoethanol. Analogous to compound 17, 2-methylpyridine derivative 18 was synthesized from 2methylnicotinoyl chloride and anthranilamide followed by cyclization, chlorination and introduction of aniline moiety. 8 ACS Paragon Plus Environment


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Figure 4: Compound synthesis scheme. Reagents and conditions were following: (i) nicotinoyl chloride hydrochloride, triethylamine, dichloromethane, tetrahydrofuran, reflux, 15 h; (ii) aqueous 1M KOH, 100 °C, 10 h; (iii) POCl3, DMF, 80 °C; (iv) HCl, dioxane, reflux, overnight; (v) isonicotinoyl chloride hydrochloride, triethylamine, dichloromethane, tetrahydrofuran, reflux, 15 h.

Two acyl derivatives 11, 12 were further subjected to reduction using sodium borohydride to give alcohols 13 and 14 (Figure 5).

Figure 5: Scheme of the synthesis of compounds 13 and 14. Reagents and conditions were as follows: (i) NaBH4, DBU, ethanol, rt, 4-6 h; (ii) HCl.


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Structure-activity relationship Quinazoline represents the parental heterocycle of the compound series. Chemical variations at the pyridine and phenyl rings of the initial hit were performed to reveal the structural features of pharmacophore 16 responsible for the binding to HIV-1 CA CTD. Furthermore, we aimed to identify binders with lower dissociation constants, to improve the solubility of the compounds and, finally, to gain detailed structure-activity relationship information that would guide further optimization cycles. The activity of compounds was assessed in the Alpha-Screen binding assay (Table 1, “IC50 AlphaScreen”), and by their potential to inhibit production of infectious HIV-1 in tissue culture (Table 1,“IC50 antiviral assay”). Cytotoxicity of compounds was assessed by a standard cell viability assay which was performed in parallel to the infectivity measurements (Table 1, cytotoxicity; see also Supplementary Figure 2 and 3 for an example data set). As evident from the data compiled in Table 1 (and similarly in Table 2 below), effects on infectivity in tissue culture did not exactly parallel CA binding potency in vitro as assessed by the AlphaScreen, indicating differences in solubility, cell permeability and bioavailability between different compounds. However, a subset of compounds abolished HIV-1 infection of TZM-bl cells at concentrations where cytotoxicity was undetectable.

The parallel preparation of two pyridine series, namely pyridine-3-yl (10-26) and pyridine-4yl (27-36), was conceived to analyze the role of pyridine in CA binding (see Tables 1&2). We hypothesized that the derivatives of pyridine-4-yl might have lower solubility in aqueous solution and thus might compromise in vitro screening. Therefore, we concentrated on the pyridine-3-yl-containing compounds. Removal of the N-3 atom of the quinazoline ring (yielding quinoline from quinazolines) substantially suppressed the activity (data not shown).



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Table 1: Structure-activity relationship of the derivatives of lead quinazoline compound 16.

IC50 (µM) (AlphaScreen)

compound R2

IC50 (µM) (antiviral assay) **

cell viability at 10 µM (%)**

10

m-COMe

30

~8

89

11

p-COMe

14

~8

93

12

p-COCF3

> 100

n.a.

62

13

p-CH(OH)Me

> 100

> 10

98

14

p-CH(OH)CF3

> 100

~10

87

15

p-C(OH)(CF3)2

> 100

n.a.

46

16

m-COOEt

68

~5

83

17

p-COOEt

47

4.7

84

18*

p-COOEt

14

> 10

100

19

p-CH2COOEt

>100

> 10

98

20

p-C(=O)NHEt

>100

> 10

97

21

p-C(=O)NH(CH2)2OH

>100

> 10

100

22

p-C(=O)NH(CH2)2F

3

> 10

90

23

p-C(=O)NHOMe

63

> 10

99

24

p-C(=O)NMeOMe

37

> 10

97

25

p-SO2NH2

80

> 10

98

26

p-SO2NHEt

>100

> 10

97

* quinazoline derivative possessing 2-methylpyridine-3-yl moiety (R1=Me) **relative to DMSO control; see Experimental Section for details n.a., non applicable

The first SAR round concentrated on carbonyl/carboxyl derivatives and their bioisosters. Phenylmethylketones such as 10 and 11 were introduced as a potential replacement of esters. The binding activity of 11 was slightly, but not significantly better than of 10. Subsequently we chose the trifluoroacetyl group, which combined a highly 11 ACS Paragon Plus Environment

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electrophilic carbonyl group with a lipophilic trifluoromethyl moiety. We hypothesized that the carbonyl group might partially exist as germinal diol (similar observation described earlier25). Our hypothesis was confirmed by NMR analyses of prepared hydrochloride of compound 12, which was shown to be capable of potential hydrogen bonding with proteins. We also hypothesized that the trifluoracetyl group would covalently react with free primary amines of CA forming an imine and resulting in covalent binding. However, derivative 12 had limited solubility in aqueous systems and its activity could not be determined. We thus turned our attention to corresponding alcohols 13, 14 that are easily accessible from ketones 11, 12. Although alcohols were not active, we were inspired by work of Li et al.26 describing the hexafluoroisopropyl group as viable moiety in a drug design which uniquely combines lipophilicity with a relatively acidic hydroxyl group. Again, screening revealed no activity of such amphiphilic alcohols. Therefore, we decided to return to the ethyl ester moiety of hit 16 and to investigate the binding of a variety of esters. Esters of 3-aminobenzoic acid and 4-aminobenzoic acid showed good binding in the AlphaScreen and also exhibited antiviral activity in tissue culture (16, 17, Table 1). These compounds (16 and 17) were relatively poorly soluble, however. Thus, we decided to lower the symmetry of the compound and to increase the dipole moment to improve the solubility of the emerging compound in aqueous solutions.27,28 We introduced a methyl group to pyridine C-2 position, which yielded a three-fold increase in binding affinity compared to 17 (18, Table 1), while this compound appeared to be less active in tissue culture. The introduction of a methylene unit in 19 between the ester group and the aromatic core of the compound led to an almost complete loss of binding affinity (19, Table 1), probably due to higher flexibility and thus unfavorable interactions of the ester moiety with the protein. Later we approached amides since they represent bioisosters of esters and also for their potentially better solubility. However, the N-ethyl amide isosteres (20) did not show any binding to CA. A further step was to employ enlarged isosteric groups with introduced hydroxy and fluorogroups (21 and 22). While the introduction of OH- group did not improve binding, the fluoro-congener 22 improved binding to CA in vitro compared to the parental compound 16. Other amides like methoxyamides (23 and 24) did not improve binding significantly. The sulfonamide bioisosteres 25 and 26 also did not show any significant in vitro activity.



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The best pyridine-3-yl derivatives of lead compound 16 thus show low micromolar activity in CA binding and antiviral activity tests and the only replacement for the ester R2 that was compatible with the activity in para or meta position was phenylmethylketone. Any other modification led to a complete loss of activity and/or solubility of the tested compound.

Table 2: Structure-activity relationship of the compounds modified in the para- position of 4N-aryl-2-(pyridine-4-yl)quinazolines. * see Experimental Section for details

compound R2

R2 HN

IC50 (µM) (AlphaScreen)

(antiviral assay)

cell viability at 10 µM (%)

*

*

IC50 (µM)

27

p-COMe

9

4.6

96

28

p-COOH

>100

> 10

98

29

p-C(=O)NH2

>100

> 10

97

30

p-CN

4

> 10

96

31

o-COOEt

37

> 10

97

32

m-COOEt

21

5.3

96

33

p-COOEt

8

> 10

96

34

p-SO2Me

>100

> 10

97

35

p-S(=O)Me

>100

> 10

98

36

p-SMe

beads aggregation

10

95

N N N

The second SAR round was based on isomeric pyridin-4-yls. Again, CA affinity as assessed by the AlphaScreen, antiviral activity and cytotoxicity were determined for all compounds from this series (Table 2). The starting compound of the series, 27, represents one of the most active compounds of the quinazoline family. The short lipophilic acetyl groups apparently contribute well to the mutual interaction between compound and protein regardless of the meta/para substitution or whether in the pyridine-3-yl or pyridine-4-yl series. 13 ACS Paragon Plus Environment

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Introduction of either acid (28) or primary amide (29) instead of the carbonyl completely prevented binding, on the other hand. A nitril group (30) caused remarkable activity in vitro but considerably decreased solubility, probably explaining lack of antiviral potency. Since ethyl esters proved favorable in the previous series they were used also in pyridine-4-yl congeners. We again started with the isomeric ethyl esters 31 and 32. The introduction of the ester into meta- or ortho- positions slightly decreased their binding in vitro in comparison to the activity of 30. The third isomer 33, having ethoxy carbonyl moiety in para position, proved to be superior in vitro, but again was inactive in tissue culture. Compound 33 provided also reliable calorimetric data enabling characterization of the thermodynamics and stoichiometry of binding (see below). The other compounds of the series were poorly soluble. They also displayed higher melting temperatures that could be explained by higher symmetry and more favorable crystal packing, both negatively influencing the dipole moment and thus solubility. The most potent compounds of the second series therefore represent the scaffold 27. The activity of the acetyl group in 27 inspired us to employ isosteric sulfon derivatives. However, neither the sulfide 36 nor sulfoxide 35 or sulfon 34 showed significant binding to CA. In conclusion, the pyridin-4-yl series also confirmed that esters of the carboxyl in the para or meta position of ring A led to low micromolar binders of CA CTD. None of the tested modifications in the R2 moiety improved binding.

Thermodynamic analysis of ligand binding to CA Isothermal titration calorimetry was used to examine binding of CAI to CA or CA CTD. CAI bound to each subunit of CA or CA CTD dimers with a large favorable enthalpic contribution (-12.5 kcal.mol-1 or -15.1 kcal.mol-1) and unfavorable entropic contribution (5.1 kcal.mol-1 or 7.6 kcal.mol-1); no protonation was observed upon binding. The dissociation constants determined by ITC (4.0 or 3.0 µM) were comparable to those measured by fluorescence polarization using fluorescently labeled CAI.22 In order to further characterize the binding of arylquinazoline compounds to HIV-1 CA or CA CTD, we monitored the binding of compound 33 by ITC (Figure 6). The considerable heat of ligand dilution was subtracted from the reaction heat by performing the control experiment of injecting ligand into buffer alone. Dimeric CA showed a 1:1 stoichiometry to 33 and a KD value of 140 ± 20 nM, the dimeric CA CTD showed a KD of 38 ± 20 nM with identical stoichiometry (Figure 6). In contrast to CAI, the binding of compound 33 was both enthalpically (-2.3 ± 0.7 kcal.mol-1 14 ACS Paragon Plus Environment


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to CA or -1.9 ± 0.5 kcal.mol-1 to CA CTD) and entropically driven (-7.1 ± 0.8 kcal.mol-1 to CA or -8.2 ± 0.8 kcal.mol-1 to CA CTD).

Figure 6: Isothermal titrations of CAI (A and B) and compound 33 (C and D) to HIV-1 CA or CA CTD. Experimental details are described in Experimental Section.

A detailed mutational analysis of CA residues mediating the tight interaction with CAI was previously described together with analysis of the effect of these mutations on HIV assembly, polyprotein processing, infectivity and morphology.22 In order to map the binding site of the compounds on the CA protein HIV-1 CA , we purified recombinant CA proteins carrying mutations Y169A, N183A, E187A, and L211A or S, respectively, which affect the CAI binding pocket.22


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Table 3: Thermodynamic parameters of CAI peptide and compound 33 binding to wild-type and mutant CAs. For experimental details see Materials and Methods. ∆G CA

∆H

- T.∆S

kcal.mol-1 kcal.mol-1 kcal.mol-1

KD

KD

µM

ratio

ligand = CAI peptide wild-type

- 7.4

- 12.5

5.1

4.0 ± 0.5

1

Y169A

- 6.7

- 12.6

6.0

13 ± 2

3

N183A

- 5.8

- 7.7

1.9

53 ± 4

13

E187A

- 7.2

- 12.3

5.1

5.0 ± 0.1

1

L211A

- 6.6

- 13.2

6.7

15 ± 3

4

L211S

- 5.6

- 4.7

- 0.8

84 ± 3

21

ligand = compound 33 wild-type

- 9.4

- 2.3

- 7.1

0.14 ± 0.02

1

N183A

- 8.3

- 3.7

- 4.6

0.89 ± 0.12

7

L211S

- 7.8

- 6.3

- 1.5

1.9 ± 0.2

14

Binding of these mutant proteins to the CAI peptide was characterized by isothermal titration calorimetry (ITC), revealing the strongest increase in KD for N183A and L211S (Table 3). These CA protein variants with the highest CAI binding defect was further analyzed with compound 33. In accordance with CAI peptide, both mutant CA proteins bound compound 33 with lower affinity than wild-type CA. The relative decrease of binding potency mirrors that observed for the CAI peptide and thus indicates that both types of inhibitors interact with the same binding pocket on the CA protein. The previous thermodynamic experiments performed with benzodiazepines and benzimidazoles targeting CA NTD showed that these inhibitors bind with submicromolar dissociation constants and are enthalpically driven.13 The other enthalpically driven CA binding inhibitor PF74 binds to the full length CA and CA NTD with low micromolar KD (2.8 µM and 2.2 µM, respectively)7 and the affinity of PF74 is 22-fold increased to hexameric CA compared with monomeric CA.18 The binding affinities of other NTD binders pyrrolopyrazolones were estimated to be low micromolar.29 The NTD-NTD interface binder



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I-XW-053 was determined as enthalpically driven ligand with binding affinity of 85 µM as measured by ITC.30 When compared with enthalpically driven inhibitors targeting NTD domain, newly identified CTD binder, arylquinazoline derivative 33, is both enthalpically and entropically driven ligand with submicromolar dissociation constant. The dominant favorable entropic contribution to the binding affinity together with chemical structure of ligand indicate that the hydrophobic interaction between CTD and 33 is largely responsible for the binding affinity. However, a structural analysis of the complex of an arylquinazoline binder with HIV CA would be necessary for the detailed description of the binding forces.

Conclusions : In conclusion, we report the identification of arylquinazolines as a novel scaffold of HIV-1 assembly inhibitors. To our knowledge, they represent the first small molecule inhibitors described that bind to the CTD of HIV-1 CA. Affinity to the target site was moderate in vitro, with low micromolar or submicromolar affinities determined by AlphaScreen competitive binding assay or ITC. However, a subset of compounds could be shown to display antiviral activity under tissue culture conditions, inhibiting the formation of infectious HIV-1 from virus producing cells. Although we could not quite rule out the possibility that the antiviral activity could result from an off-target effect on the virus, The determined in vitro binding characteristics indicate that the small molecules described here target the same binding pocked as the previously described CAI peptide.20-21 In the case of the CAI peptide, disruption of a critical interface in the mature lattice, formed between the CAI binding pocket located in the CTD with the NTD of the same CA molecule, appears to be the major mode of mature assembly inhibition in vitro22, and the observation that the newly described inhibitors target the same site suggests that inhibition of mature capsid assembly is a relevant mechanism of action. However, CAI was also found to inhibit assembly of immature-like particles in vitro by a yet unknown mode of action. Thus, both impaired particle assembly and impaired formation of the mature, infectious core might contribute to the observed antiviral activity of arylquinazoline derivatives; the relative quantitative importance of these mechanisms, however, awaits careful dissection in future studies.


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Experimental Section :

Chemical methods All commercial chemicals and solvents were reagent grade and used without further purification unless otherwise specified. All reactions except those in aqueous media were carried out under an argon atmosphere or with the use of standard techniques for exclusion of moisture. Reactions were monitored by thin-layer chromatography on silica gel plates (Merck Kieselgel 60 F254) and visualized with UV, iodine vapours, ninhydrin and Seebach’s stain. Purification by column chromatography was typically performed using silica gel 60 (Merck) and solvent mixtures and gradients are recorded herein. Standard workup procedures were used for all reactions and combined organic layers were dried over MgSO4 before evaporation. 1H and 13C NMR spectra were recorded using Brucker 400 MHz and 600 MHz instruments. The chemicals shifts are reported in parts per million (ppm) based on the reference of the deuterated solvent. Mass spectra (HRMS) measurements were recorded on LTQ Orbitrap XL and the mass ion was determined by electrospray ionization. Melting points were measured on Kofler apparatus and are uncorrected. IR spectra were recorded on PerkinElmer FT-IR or Bruker Alpha-P FT-IR spectrophotometer. Purity of the prepared compounds was checked by HPLC chromatography by using Agilent Technologies HPLC-MS system (1260 Infinity HPLC with 6530 Accurate Mass Q TOF) with a binary solvent system. All final compounds had ≥95% purity. The analyses were carried out under the following conditions: Zorbax Extended-C18-600 Bar column (2.1 × 50 mm; 1.8 Micron); mobile phase, A = H2O with 0.1% formic acid and B = CH3CN; gradient: 5–100% B (0.0–15.0 min); UV detection at 254 nm. Detailed experimental procedures of the intermediates are described in the Supporting Information. Data bellow are for the final compounds tested in the assay. General procedure A. A mixture of a 4-chloroquinazoline derivative (1.0 eq.), aryl amine (1.0 eq.) and HCl (two drops of aqueous HCl or indicated amount of 4M HCl in dioxane) in dioxane was stirred at 100 °C overnight. The mixture was allowed to cool down to room temperature and the formed precipitate was collected by suction filtration. Recrystallization of the precipitate cake from MeOH furnished the desired product. General procedure B. A mixture of starting quinazoline derivative (1.0 eq.), aryl amine (1.0 eq.) and HCl (1.0 eq., solution in dioxane) in dioxane was stirred at 100 °C overnight. The mixture was allowed to cool to room temperature and was diluted with H2O. Then aqueous 18 ACS Paragon Plus Environment


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NH3 and NaHCO3 (saturated solution) was added stepwise. The product was extracted with DCM, the combined organic layers were washed with water, brine, dried over MgSO4 and concentrated under reduced pressure. Column chromatography of the residue on silica gel afforded the product as a free base that was further converted to the corresponding hydrochloride salt by treatment with 4M HCl in dioxane. N-(3-Acetylphenyl)-2-(pyridin-3-yl)quinazolin-4-amine

hydrochloride

(10).

The

title

compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.30 g, 1.24 mmol), 3aminoacetophenone (0.17 g, 1.24 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 0.31 g (61%) of the title compound as a colourless solid. Salt: mp 199-202 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.53 (d, J = 1.7 Hz, 1H), 9.21 (d, J = 8.1 Hz, 1H), 8.99 (d, J = 5.3 Hz, 1H), 8.93 (d, J = 8.3 Hz, 1H), 8.59 (s, 1H), 8.22-8.18 (m, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.05 (dd, J = 7.9, 5.4 Hz, 1H), 8.00 (t, J = 7.7 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 2.63 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 199.3, 160.3, 156.3, 147.7, 145.5, 143.5, 140.1, 138.9, 136.6, 135.3, 130.7, 129.6, 129.5, 127.9, 126.7, 126.6, 125.9, 124.4, 115.6, 28.6. IR (KBr) ν 3433, 3026, 2788, 1678, 1634, 1593, 1453, 1419, 1363, 1274 cm-1. HR-MS (ESI) calcd. for C21H17ON4+ [M+H+] 341.1397, found 341.1396. N-(4-Acetylphenyl)-2-(pyridin-3-yl)quinazolin-4-amine

hydrochloride

(11).

The

title

compound was prepared from 4-chloro-2-(pyridin-3-yl)-quinazoline 4 (0.7 g, 2.89 mmol), 4aminoacetophenone (0.39 g, 2.89 mmol) and HCl (0.7 mL of 4 M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 1.03 g (86%) of the title compound as a colourless solid. Salt: mp 226-229 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.93 (bs, 1H), 9.55 (d, J = 1.5 Hz, 1H), 9.22 (d, J = 8.1 Hz, 1H), 9.02 (d, J = 4.7 Hz, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.36-8.10 (m, 3H), 8.08 (m, 3H), 7.98 (dd, J = 11.4, 4.0 Hz, 1H), 7.74 (dd, J = 11.2, 4.0 Hz, 1H), 2.61 (s, 3H).

13

C NMR (100 MHz, DMSO-d6) δ

196.7, 158.3, 154.6, 147.6, 144.9, 143.1, 142.8, 142.2, 134.6, 132.6, 129.2, 129.0, 127.8, 126.6, 126.3, 124.1, 122.1, 114.2, 26.6. IR (KBr) ν 3273, 3071, 2491, 1632, 1615, 1553, 1492, 1434, 1367, 1318, 1157, 1094 cm-1. HR-MS (ESI) calcd. for C21H17ON4+ [M+H+] 341.1397, found 341.1397. N-(4-Trifluoroacetylphenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride (12). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.30 g, 1.24 mmol), 419 ACS Paragon Plus Environment

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(trifluoroacetyl)aniline (0.24 g, 1.24 mmol) and HCl (0.3 mL of 4 M solution in dioxane) according to the general procedure B. Column chromatography on silica gel (EtOAc) yielded 0.39 g (75%) of the free base that was converted to hydrochloride salt 12 by treatment with HCl. Base: 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.61 (d, J = 1.5 Hz, 1H), 8.88-8.71 (m, 2H), 8.67 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 9.0 Hz, 2H), 8.21 (d, J = 8.2 Hz, 2H), 7.98 (d, J = 3.8 Hz, 2H), 7.81-7.68 (m, 1H), 7.61 (dd, J = 7.9, 4.8 Hz, 1H). Salt: mp 273-275 °C (MeOH). 1

H NMR (400 MHz, DMSO-d6) δ 10.63 (bs, 1H), 9.62 (d, J = 1.6 Hz, 1H), 9.03 (m, 1H), 8.88

(dd, J = 5.1, 1.4 Hz, 1H), 8.74 (t, J = 6.8 Hz, 1H), 8.38 (d, J = 9.0 Hz, 2H), 8.21 (d, J = 8.2 Hz, 2H), 8.01 (d, J = 3.8 Hz, 3H), 7.87 (dd, J = 8.0, 5.2 Hz, 1H), 7.77 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ complex spectrum due to dynamic formation of heminal. IR (KBr) ν 3432, 3051, 2686, 1628, 1603, 1547, 1456, 1433, 1409, 1177, 1164 cm-1. HR-MS (ESI) calcd. for C21H14ON4F3+ [M+H+] 395.1114, found 395.1111. N-(4-(1-Hydroxyethyl)phenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride (13). To a suspension of N-(4-acetylphenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride 11 (0.3 g, 0.8 mmol), DBU (0.12 g, 0.8 mmol) in EtOH/THF (11 mL, v/v) was added portionwise NaBH4 (0.06 g, 1.6 mmol). The reaction mixture was stirred for 4h, then it was quenched with H2O, and extracted with EtOAc (3×10 mL), the combined organic layers were washed with H2O, brine, dried over Na2SO4, and concentrated under reduced pressure. Two-fold column chromatography on silica gel (10/1 DCM/MeOH and 10/1 EtOAc/MeOH) furnished 0.19 g (69%) of the title compound as free base that was converted to corresponding hydrochloride salt by treatment with 4M HCl in dioxane. Base: 1H NMR (400 MHz, DMSO-d6) δ 9.95 (bs, 1H), 9.54 (m, 1H), 8.77-8.66 (m, 2H), 8.61 (d, J = 8.4 Hz, 1H), 7.89 (m, 4H), 7.66 (dt, J = 12.1, 3.9 Hz, 1H), 7.58 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 5.19 (d, J = 4.2 Hz, 1H), 4.76 (m, 1H), 1.39 (d, J = 6.4 Hz, 3H). Salt: mp 186-188 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.08 (bs, 1H), 9.54 (d, J = 1.4 Hz, 1H), 8.75 (m, 2H), 8.63 (d, J = 8.4 Hz, 1H), 7.90 (m, 3H), 7.74-7.57 (m, 2H), 7.46 (d, J = 8.4 Hz, 2H), 4.79 (q, J = 6.4 Hz, 1H), 1.39 (d, J = 6.4 Hz, 3H).

13

C NMR (100 MHz,

DMSO-d6) δ 158.5, 157.6, 150.8, 149.9, 149.1, 143.6, 137.7, 136.3, 134.1, 133.9, 127.9, 126.9, 125.9, 124.3, 123.7, 122.8, 114.6, 68.3, 26.1. IR (KBr) ν 3385, 2649, 2610, 1627, 1604, 1549, 1515, 1457, 1437, 1375, 1360, 1158, 1090 cm-1. HR-MS (ESI) calcd. for C21H19ON4+ [M+H+] 343.1553, found 343.1553. 20 ACS Paragon Plus Environment


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N-(4-(2,2,2-Trifluor1-hydroxyethyl)phenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride (14). To the suspension of N-(4-trifluoroacetylphenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride 12 (0.31 g, 0.72 mmol), DBU (0.11 g, 0.72 mmol) in EtOH (10 mL) was added portionwise NaBH4 (0.03 g, 0.72 mmol). The reaction mixture was stirred for 6h and then the reaction was quenched with H2O and extracted with DCM. The combined organic layers were washed with water, brine, dried over Na2SO4 and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (10/1 DCM/MeOH) to give 0.20 g (70%) of the title compound as free base that was converted to the corresponding hydrochloride salt by treatment with 4M HCl in dioxane. Base: 1H NMR (400 MHz, DMSOd6) δ 10.04 (s, 1H), 9.53 (m, 1H), 8.76-8.68 (m, 2H), 8.62 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 3.7 Hz, 2H), 7.68 (dt, J = 8.3, 4.2 Hz, 1H), 7.60 (dd, J = 6.3, 4.7 Hz, 2H), 7.58 (m, 1H), 6.87 (d, J = 5.5 Hz, 1H), 5.19 (m, 1H). Salt: mp 247250 °C (MeOH). 1H NMR (100 MHz, DMSO-d6) δ10.79 (bs, 1H), 9.53 (d, J = 1.7 Hz, 1H), 9.10 (d, J = 8.1 Hz, 1H), 8.96 (dd, J = 5.3, 1.4 Hz, 1H), 8.82 (d, J = 8.2 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.07-7.92 (m, 3H), 7.81-7.72 (m, 1H), 7.63 (d, J = 8.5 Hz, 2H), 5.24 (q, J = 7.3 Hz, 1H). Salt:

13

C NMR (150 MHz, DMSO-d6) δ 159.2, 155.5, 147.4, 146.3, 145.1, 141.9,

138.9, 135.5, 133.9, 133.2, 128.6, 128.5, 126.7, 126.7, 125.5, 124.9, 123.7, 114.5, 70.8 (JC-F = 30.4 Hz). IR (KBr) ν 3058, 2698, 1631, 1607, 1550, 1437, 1264, 1162, 1125 cm-1. HR-MS (ESI) calcd. for C21H16ON4F3+ [M+H+] 397.1270, found 397.1270. 1,1,1,3,3,3-Hexafluoro-2-[4-[2-(pyridin-3-yl)-quinazolin-4-yl]amino]phenyl]propan-2-ol hydrochloride (15). The title compound was prepared from 4-chloro-2-(pyridin-3yl)quinazoline

4

(0.30

g,

1.24

mmol),

4-[2,2,2-trifluoro-1-hydroxy-1-

(trifluoromethyl)ethyl]phenylamine (0.32 g, 1.24 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from i-PrOH/MeOH yielded 0.32 g (51%) of the title compound as a colourless solid. Salt: mp 217-220 °C (iPrOH/MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.74 (bs, 1H), 9.57 (d, J = 1.7 Hz, 1H), 9.17 (d, J = 8.2 Hz, 1H), 8.98 (dd, J = 5.4, 1.4 Hz, 1H), 8.85 (d, J = 8.2 Hz, 1H), 8.80 (s, 1H), 8.19 (d, J = 9.0 Hz, 2H), 8.11-7.97 (m, 3H), 7.88-7.80 (m, 2H), 7.75 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 159.1, 155.7, 147.9, 146.7, 144.8, 142.1, 140.7, 135.3, 134.8, 128.4, 127.9, 127.1, 126.8, 126.7, 124.7, 124.1 (JC-F = 288 Hz), 123.2, 114.7, 77.5 (JC-F = 30 Hz). IR (KBr) ν 3405, 3076, 2665, 1630, 1604, 1547, 1519, 1435, 1377, 1270, 1208, 1189 cm-1. HRMS (ESI) calcd. for C22H13ON4F6+ [M+H+] 463.0988, found 463.0996.


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N-(3-(Ethoxycarbonyl)phenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride (16). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.30 g, 1.24 mmol), ethyl 3-aminobenzoate (0.20 g, 1.24 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 0.31 g (57%) of the title compound as a pale yellow solid. Salt: mp 194-196 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 11.03 (bs, 1H), 9.57 (d, J = 1.8 Hz, 1H), 9.19 (d, J = 8.2 Hz, 1H), 9.00 (dd, J = 5.4, 1.4 Hz, 1H), 8.88 (d, J = 8.1 Hz, 1H), 8.68 (t, J = 1.8 Hz, 1H), 8.24 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.04 (ddd, J = 8.2, 7.6, 3.1 Hz, 2H), 7.88 (m, 1H), 7.79 (m, 1H), 7.66 (t, J = 7.9 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). 13

C NMR (100 MHz, DMSO-d6) δ 166.2, 159.2, 155.3, 146.8, 146.6, 144.6, 142.3, 139.0,

135.6, 134.1, 130.9, 129.7, 128.6, 128.4, 126.9, 126.2, 125.7, 124.9, 124.4, 114.5, 61.7, 14.9. IR (KBr) ν 3436, 3040, 2439, 1706, 1635, 1549, 1467, 1455, 1383, 1292, 1278, 1022 cm-1. HR-MS (ESI) calcd. for C22H19O2N4+ [M+H+] 371.1503, found 371.1502. N-(4-(Ethoxycarbonyl)phenyl)-2-(pyridin-3-yl)quinazolin-4-amine hydrochloride (17). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.25 g, 1.03 mmol), ethyl 4-aminobenzoate (0.34 g, 2.06 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 0.31 g (74%) of the title compound as a pale yellow solid. Salt: mp 215-216 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 10.64 (bs, 1H), 9.58 (s, 1H), 9.14 (s, 1H), 8.96 (d, J = 4.2 Hz, 1H), 8.78 (d, J = 8.3 Hz, 1H), 8.17 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.8 Hz, 2H), 8.06-7.92 (m, 3H), 7.75 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSOd6) δ 165.9, 158.6, 155.5, 149.1, 146.1, 144.3, 143.5, 141.7, 135.4, 134.9, 130.3, 128.1, 127.6, 126.7, 125.4, 124.0, 122.3, 114.7, 61.1, 14.7; IR (KBr) ν 3425, 3056, 2545, 1711, 1605, 1569, 1524, 1411, 1277, 1175, 1106, 1020 cm-1. HR-MS (ESI) calcd. for C22H19O2N4+ [M+H+] 371.1503, found 371.1503. N-(4-(Ethoxycarbonyl)phenyl)-2-(2-methylpyridin-3-yl)quinazolin-4-amine

hydrochloride

(18). The reaction of 4-chloro-2-(2-methylpyridin-3-yl)quinazoline 6 (0.18 g, 0.71 mmol) and ethyl 4-aminobenzoate (0.12 g, 0.75 mmol) according to general procedure A furnished 0.23 g (84 %) of the title compound as a yellow solid. Salt: mp 156-160 °C (MeOH). 1H NMR (500 MHz, DMSO-d6) δ 10.89 (bs, 1H, NH), 8.96 (dd, J = 8.0, 1.6 Hz, 1H), 8.86 (dd, J = 5.7, 1.6 Hz, 1H), 8.83 (m, 1H), 8.00-8.04 (m, 7H), 4.31 (q, J = 7.1 Hz, 2H), 2.99 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H).

13

C NMR (125 MHz, DMSO-d6) δ 165.7, 158.7, 157.0, 154.0, 147.8, 146.1,

143.1, 142.9, 135.6, 135.0, 130.2, 128.4, 126.6, 125.8, 124.7, 124.2, 123.0, 113.9, 61.0, 20.3, 22 ACS Paragon Plus Environment


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14.5. IR (ATR) 3762, 3661, 1722, 1658, 1546, 1546 cm-1. HR-MS (ESI) calcd. for C26H27N4O4+ [M+H+] 385.1659; found, 385.1658. Ethyl 4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-phenylacetate hydrochloride (19). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.5 g, 2.07 mmol), ethyl (4-aminophenyl)acetate (0.37 g, 2.07 mmol) and HCl (0.5 mL of 4 M solution in dioxane) according to the general procedure A. recrystallization from EtOH yielded 0.63 g (66%) of the title compound as a colourless solid. Salt: mp 178-180 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 11.15 (bs, 1H), 9.52 (d, J = 1.8 Hz, 1H), 9.12 (d, J = 8.1 Hz, 1H), 8.98 (dd, J = 5.3, 1.4 Hz, 1H), 8.92 (d, J = 8.1 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 8.08-7.95 (m, 3H), 7.87 (d, J = 8.5 Hz, 2H), 7.77 (m, 1H), 7.41 (d, J = 8.5 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.74 (s, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 172.7, 158.5, 154.4, 146.6, 144.3, 143.9, 141.8, 135.9, 135.2, 132.4, 132.3, 129.6, 128.1, 126.1, 124.3, 123.6, 132.5, 113.4, 56.1, 40.3, 18.5; IR (KBr) ν 3429, 2957, 2740, 1984, 1725, 1632, 1552, 1436, 1385, 1368, 1334, 1259, 1140 cm-1. HR-MS (ESI) calcd. for C23H21O2N4+ [M+H+] 385.1659, found 385.1659. N-Ethyl-4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzamide hydrochloride (20). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 6 (0.24 g, 1.46 mmol), 4amino-N-ethylbenzamide (0.35 g, 1.46 mmol) and HCl (0.36 mL of 4 M solution in dioxane) according to the general procedure A. Crystallization from EtOH followed by recrystallization from MeOH yielded 0.22 g (34%) of the title compound as a colourless solid. Salt: mp 180183 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 11.05 (bs, 1H), 9.55 (d, J = 1.6 Hz, 1H), 9.19 (d, J = 8.2 Hz, 1H), 9.01 (dd, J = 5.3, 1.0 Hz, 1H), 8.91 (d, J = 8.2 Hz, 1H), 8.58 (t, J = 5.4 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.10-7.94 (m, 5H), 7.76 (m, 1H), 3.43-3.15 (d, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 165.8, 158.8, 154.4,

144.6, 143.3, 140.5, 135.8, 133.4, 131.6, 128.5, 128.1, 127.0, 127.0, 125.0, 123.3, 114.1, 34.5, 15.3; IR (KBr) ν 3427, 2598, 1632, 1546, 1496, 1433, 1389, 764 cm-1. HR-MS (ESI) [M+H+] calcd. for C22H20ON5+ [M+H+] 370.1662, found 370.1662. N-(2-Hydroxyethyl)-4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzamide

hydrochloride

(21). N-(4-(Ethoxycarbonyl)phenyl)-2-(pyridin-3-yl)quinazoline-4-amine 17 (0.22 g; 0.54 mmol) was dissolved in ethanolamine (10 mL, 165.7 mmol) and the reaction mixture was stirred at 80 °C for 36 h. The reaction mixture was allowed to cool down to room temperature and then it was concentrated under reduced pressure. Column chromatography of the residue 23 ACS Paragon Plus Environment

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on silica gel (10/1 CH2Cl2/MeOH) furnished the crude product as free base that was further converted to hydrochloride salt by treatment with HCl (1M aqueous solution). Recrystallization from MeOH yielded 0.18 g (78%) of the title compound as a yellow solid. Salt: mp 162-165 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 1H NMR (401 MHz, DMSO-d6) δ 11.02 (s, 1H), 9.55 (s, 1H), 9.19 (d, J = 8.1 Hz, 1H), 9.01 (bs, 1H), 8.88 (d, J = 8.3 Hz, 1H), 8.57 (t, J = 5.4 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.09 (m, 1H), 7.99 – 8.05 (m, 5H), 7.78 (t, J = 7.6 Hz, 1H), 3.54 (t, J = 6.2 Hz, 2H), 3.36 (dd, J = 6.0 Hz, 2H). 13C NMR: (101 MHz, DMSO-d6) δ 165.9, 158.6, 154.8, 143.8, 141.9, 140.7, 140.7, 135.0, 128.0, 127.9, 126.5, 125.7, 124.6, 122.7, 122.6, 114.1, 59.9, 42.3, 39.5. FT–IR (CHCl3) ν 3429, 2939, 2802, 2678, 1635, 1546, 1433 cm-1. HR-MS (ESI) calcd. for C22H20N5O2+ [M+H+] 386.16115; found 386.16117. N-(2-Fluoroethyl)-4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzamide

hydrochloride

(22). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.30 g, 1.24 mmol), 4-amino-N-(2-fluoroethyl)benzamide (0.22 g, 1.24 mmol) and HCl (0.3 mL of 4 M solution in dioxane) according to the general procedure A. Recrystallization from iPrOH/MeOH yielded 0.22 g (39%) of the title compounds as a pale yellow solid. Salt: mp 296-298 °C (i-PrOH/MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.51 (bs, 1H), 9.56 (s, 1H), 8.93 (d, J = 8.1 Hz, 1H), 8.85 (m, 1H), 8.76 (t, J = 5.9 Hz, 3H), 8.14-8.07 (m, 2H), 8.06-7.91 (m, 4H), 7.81 (dd, J = 8.0, 5.1 Hz, 1H), 7.72 (ddd, J = 8.2, 6.5, 1.7 Hz, 2H), 4.59 (m, 2H), 3.73 – 3.55 (m, 2H).

13

C NMR (100 MHz, DMSO-d6) δ 166.7, 159.1, 155.3, 147.3, 146.5,

144.4, 142.4, 141.5, 135.4, 134.6, 130.9, 128.5, 127.0, 126.2, 124.8, 123.2, 122.3, 114.7, 82.8 (JC-F = 165 Hz), 40.5. IR (KBr) ν 3258, 2604, 1637, 1606, 1549, 1497, 1434, 1383, 1306, 1118, 1017 cm-1. HR-MS (ESI) calcd. for C22H19ON5F+ [M+H+] 388.1568, found 388.1568. N-Methoxy-4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzamide hydrochloride (23). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.30 g, 1.26 mmol), N-methoxy-4-aminobenzamide (0.21 g, 1.26 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure B. Column chromatography on silica gel (10/1 DCM/MeOH) furnished 0.26 g (50%) of the free base that was converted to hydrochloride salt by treatment with HCl. Salt: mp 183-185 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (bs, 1H), 10.73 (bs, 1H), 9.56 (s, 1H), 9.11 (d, J = 8.1 Hz, 1H), 8.96 (d, J = 4.5 Hz, 1H), 8.80 (d, J = 8.3 Hz, 1H), 8.06 (m, 3H), 8.01 (dd, J = 12.9, 6.0 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H), 7.83-7.66 (m, 2H), 3.75 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 63.8, 158.4, 154.7, 147.1, 145.7, 143.7, 141.8, 141.3, 134.9, 134.2, 130.3, 128.2, 127.8, 126.5, 126.0, 124.3, 24 ACS Paragon Plus Environment


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122.5, 114.1, 63.5. IR (KBr) ν 3427, 3059, 1631, 1603, 1568, 1518, 1495, 1414, 1378, 1318, 1239, 1040 cm-1. HR-MS (ESI) calcd. for C21H18O2N5+ [M+H+] 372.1455, found 372.1455. N-Methoxy-N-methyl-4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzamide

hydrochloride

(24). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)-quinazoline 4 (0.50 g, 2.07 mmol), N-methoxy-N-methyl-4-aminobenzenecarboxamide (0.37 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure B. Column chromatography on silica gel (25/1 EtOAc/MeOH) yielded 0.23 g (29%) of the free base that was converted to the corresponding hydrochloride salt by treatment with HCl. Base: 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.57 (dd, J = 2.0, 0.7 Hz, 1H), 8.71 (m, 2H), 8.63 (d, J = 8.4 Hz, 1H), 8.14-8.03 (m, 2H), 7.93 (d, J = 3.7 Hz, 2H), 7.83-7.75 (m, 2H), 7.69 (dt, J = 8.3, 4.1 Hz, 1H), 7.57 (ddd, J = 7.8, 4.9, 0.8 Hz, 1H), 3.35 (s, 3H), 3.30 (s, 3H). Salt: mp 172174 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 9.56 (d, J = 1.7 Hz, 1H), 9.20 (d, J = 8.1 Hz, 1H), 9.00 (dd, J = 5.3, 1.2 Hz, 2H), 8.87 (d, J = 8.1 Hz, 2H), 8.27-7.94 (m, 5H), 7.91-7.72 (m, 3H), 3.63 (s, 3H), 3.32 (s, 3H).

13


169.3, 159.2, 155.2, 147.1, 146.2, 144.2, 142.7, 140.8, 135.5, 134.6, 130.9, 129.2, 128.5, 127.1, 126.1, 124.9, 123.1, 114.7, 61.5, 34.1. IR (KBr) ν 3433, 3060, 2532, 1637, 1622, 1601, 1546, 1499, 1438, 1383, 1368, 1162 cm-1. HR-MS (ESI) calcd. for C22H20O2N5+ [M+H+] 386.1611, found 386.1611. 4-[[2-(Pyridin-3-yl)-quinazolin-4-yl]amino]-benzenesulfonamide hydrochloride (25). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.3 g, 1.24 mmol), 4-aminobenzenesulfonamide (0.21 g, 1.24 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 0.38 g (68%) of the title compound as colourless solid. Salt: mp >300 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 9.52 (d, J = 1.6 Hz, 1H), 9.19 (d, J = 8.1 Hz, 1H), 8.99 (dd, J = 5.3, 1.0 Hz, 1H), 8.87 (d, J = 8.3 Hz, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.12-8.03 (m, 2H), 7.97 (dd, J = 11.3, 4.0 Hz, 1H), 7.95-7.88 (m, 2H), 7.74 (m, 1H), 7.39 (bs, 2H).

13

C NMR

(100 MHz, DMSO-d6) δ 160.1, 156.5, 149.3, 147.0, 145.2, 143.7, 143.1, 141.2, 136.4, 136.3, 129.5, 128.2, 128.2, 128.1, 125.8, 124.3, 115.9. IR (KBr) ν 3369, 3289, 3027, 2492, 1634, 1618, 1565, 1548, 1521, 1495, 1420, 1385, 1337, 1158 cm-1. HR-MS (ESI) calcd. for C19H16O2N5S+ [M+H+] 378.1019, found 378.1020. N-Ethyl 4-[[2-(pyridin-3-yl)-quinazolin-4-yl]amino]-benzenesulfonamide hydrochloride (26). The title compound was prepared from 4-chloro-2-(pyridin-3-yl)quinazoline 4 (0.3 g, 1.24 25 ACS Paragon Plus Environment

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mmol), 4-amino-N-ethylbenzenesulfonamide (0.25 g, 1.24 mmol) and HCl (0.3 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from EtOH yielded 0.40 g (67%) of the title compound as a colourless solid. Salt: mp 264-270 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ 10.98 (bs, 1H), 9.57 (d, J = 1.6 Hz, 1H), 9.26 (d, J = 8.1 Hz, 1H), 9.02 (m, 1H), 8.91 (d, J = 8.3 Hz, 1H), 8.23 (d, J = 8.8 Hz, 2H), 8.19-8.06 (m, 2H), 8.02 (t, J = 7.7 Hz, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.77 (dd, J = 11.2, 4.1 Hz, 1H), 7.62 (s, 1H), 2.85 (q, J = 7.0 Hz, 2H), 1.02 (t, J = 7.0 Hz, 3H).

13


159.1, 155.4, 148.6, 145.8, 143.9, 142.8, 142.7, 136.4, 135.5, 135.3, 128.4, 128.0, 127.5, 127.3, 124.7, 123.3, 114.9, 38.3, 15.5. IR (KBr) ν 3273, 3071,2491, 1632, 1615, 1553, 1492, 1434, 1367, 1318, 1157, 1094 cm-1. HR-MS (ESI) calcd. for C21H20O2N5S+ [M+H+] 406.1332, found 406.1333. N-(4-Acetylphenyl)-2-(pyridin-4-yl)quinazolin-4-amine

hydrochloride

(27).

The

title

compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.50 g, 2.07 mmol), 4aminoacetophenone (0.28 g, 2.08 mmol) and conc. HCl (two drops) according to the general procedure B. Column chromatography on silica gel (10/1 DCM/MeOH) furnished 0.40 g (57%) of the free base that was converted to the corresponding hydrochloride salt by treatment with 4M HCl in dioxane. Base: 1H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.67 (dd, J = 4.5, 1.6 Hz, 2H), 8.53 (d, J = 8.4 Hz, 1H), 8.20 (dd, J = 4.5, 1.6 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.89-7.79 (m, 2H), 7.61 (ddd, J = 8.3, 5.0, 3.2 Hz, 1H), 3.26 (s, 3H). Salt: mp 296-297 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.02 (d, J = 6.6 Hz, 2H), 8.79 (d, J = 8.4 Hz, 1H), 8.74 (d, J = 6.6 Hz, 2H), 8.17 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.8 Hz, 2H), 8.06-7.95 (m, 2H), 7.79 (ddd, J = 8.3, 5.5, 2.7 Hz, 1H), 2.61 (s, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 197.0, 158.6, 155.3, 152.0, 150.0, 144.3,

143.8, 134.6, 132.8, 129.5, 128.8, 128.5, 124.7, 124.2, 121.9, 115.2, 26.9. IR (KBr) ν 3431, 3066, 2365, 2082, 2003, 1674, 1634, 1596, 1568, 1513, 1416, 1362, 1330, 1271, 1184 cm-1. HR-MS (ESI) calcd. for C17H17ON4+ [M+H+] 341.1397, found 341.1396. 4-[[2-(Pyridin-4-yl)-quinazolin-4-yl]amino]-benzoic acid hydrochloride (28). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.5 g, 2.07 mmol), 4aminobenzoic acid (0.28 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.37 g (47%) of the title compound as a colourless solid. Salt: mp >300 °C (MeOH). 1H NMR (400 MHz, CDCl3) δ 10.57 (s, 1H), 9.10-8.95 (m, 2H), 8.79 (d, J = 8.4 Hz, 1H), 8.72 (dd, J = 5.4, 1.3 Hz, 2H), 8.11 (d, J = 8.9 Hz, 2H), 8.05 (d, J = 8.9 Hz, 2H), 8.02-7.88 (m, 2H), 7.75 (ddd, J = 8.3, 26 ACS Paragon Plus Environment


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5.0, 3.2 Hz, 1H), 4.23 (bs, 1H).

13

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C NMR (100 MHz, CDCl3) δ 167.4, 158.7, 155.3, 152.3,

150.0, 144.1, 143.4, 134.7, 130.5, 128.8, 128.6, 126.3, 124.8, 124.2, 122.2, 115.2. IR (KBr) ν 3429, 3061, 2611, 1710, 1635, 1604, 1564, 1513, 1415, 1375, 1331, 1221, 1174, 1113 cm-1. HR-MS (ESI) [M+H+] calcd. for C20H15N4O2+ [M+H+] 343.1190, found 343.1188. 4-[[2-(Pyridin-4-yl)-quinazolin-4-yl]amino]-benzamide

hydrochloride

(29).

The

title

compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.50 g, 2.07 mmol), 4amino-benzamide (0.28 g, 2.08 mmol) and HCl (0.39 mL of 4M solution in dioxane, 1.56 mmol) according to the general procedure B. Column chromatography on silica gel (10/1 DCM/MeOH) furnished 0.57 g (80%) of the free base that was converted to the corresponding hydrochloride salt by treatment with HCl. Salt: mp >300 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.45 (bs, 1H), 9.03-8.94 (m, 2H), 8.77 (d, J = 8.4 Hz, 1H), 8.72-8.60 (m, 2H), 8.13-7.96 (m, 6H), 7.79 (ddd, J = 8.3, 5.8, 2.4 Hz, 1H), 7.35 (bs, 1H). 13C NMR (100 MHz, DMSO-d6) δ 167.9, 158.7, 156.1, 150.1, 150.0, 146.3, 142.0, 134.4, 129.9, 128.7, 128.6, 128.1, 124.1, 123.8, 122.0, 115.0. IR (KBr) ν 3464, 2739, 1657, 1569, 1526, 1511, 1415, 1375, 1332, 842 cm-1. HR-MS (ESI) calcd. for C20H16ON5+ [M+H+] 342.1349, found 342.1348. 4-[[2-(Pyridin-4-yl)-quinazolin-4-yl]amino]-benzonitrile hydrochloride (30).

The title

compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.5 g, 2.07 mmol), 4aminobenzonitrile (0.25 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.70 g (93%) of the title compound as a yellow solid. Salt: mp >300 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.45 (bs, 1H), 8.83 (m, 2H), 8.61 (m, 1H), 8.51 (m, 2H), 8.08 (m, 2H), 7.82 (m, 4H), 7.64 (m, 1H).

13

C NMR (150 MHz, DMSO-d6) δ 158.1, 155.4, 150.0, 149.9, 145.5, 143.4, 134.2,

132.9, 128.5, 128.0, 123.6, 122.2, 119.2, 114.7, 105.3. IR (KBr) ν 3430, 2487, 2218, 1634, 1603, 1570, 1511, 1413, 1373, 1331, 1246, 1180 cm-1. HR-MS (ESI) [M+H+] calcd. for C20H14N5+ [M+H+] 324.1244, found 324.1243. N-(2-(Ethoxycarbonyl)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (31). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.5 g, 2.07 mmol), ethyl 2-aminobenzoate (0.34 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.44 g (52%) of the title compound as a pale yellow solid. Salt: mp 218-219 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 9.01 (d, J = 6.3 Hz, 2H), 8.64 (d, J = 6.6 Hz, 2H), 8.45 (d, J = 8.3 27 ACS Paragon Plus Environment

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Hz, 1H), 8.34 (dd, J = 8.2, 0.8 Hz, 1H), 8.09-7.94 (m, 3H), 7.84 (m, 1H), 7.77 (ddd, J = 8.3, 7.5, 1.6 Hz, 1H), 7.38 (td, J = 7.8, 1.1 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 1.02 (t, J = 7.1 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 167.3, 158.3, 155.4, 150.1, 149.5, 145.2, 142.8,

138.9, 134.1, 133.5, 130.5, 128.5, 128.3, 124.2, 124.1, 123.6, 122.4, 114.6, 61.0, 13.6. IR (KBr) ν 3430, 3064, 2396, 2070, 1676, 1621, 1603, 1568, 1536, 1509, 1448, 1415, 1367, 1257, 1163 cm-1. HR-MS (ESI) [M+H+] calcd. for C22H19N4O2+ [M+H+] 371.1503, found 371.1501. N-(3-(Ethoxycarbonyl)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (32). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.5 g, 2.07 mmol), ethyl 3-aminobenzoate (0.34 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.68 g (81%) of the title compound as a pale yellow solid. Salt: mp 229-231 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.99 (dd, J = 5.4, 1.2 Hz, 2H), 8.77 (d, J = 8.4 Hz, 1H), 8.70 (m, 3H), 8.22 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.97 (m, 2H), 7.80 (m, 1H), 7.75 (ddd, J = 8.3, 5.5, 2.8 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.1, 158.7, 155.4, 151.7, 149.8, 144.6, 139.6, 134.6, 130.7, 129.4, 128.6, 128.5, 127.3, 125.1, 124.5, 124.1, 123.4, 115.0, 61.4, 14.7. IR (KBr) ν 3416, 2988, 2568, 1724, 1635, 1572, 1513, 1441, 1384, 1295, 1283, 1234, 749 cm-1. HR-MS (ESI) [M+H+] calcd. for C22H19N4O2+ [M+H+] 371.1503, found 371.1499. N-(4-(Ethoxycarbonyl)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (33). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)-quinazoline 9 (0.4 g, 1.65 mmol), ethyl 4-aminobenzoate (0.55 g, 3.31 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.56 g (83%) of the title compound as a pale yellow solid. Salt: mp 235-238 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.57 (bs, 1H), 9.02 (d, J = 6.6, 2H), 8.80 (d, J = 8.4, 1H), 8.75 (d, J = 6.6, 2H), 8.18 (d, J = 8.8, 2H), 8.08 (d, J = 8.8, 2H), 8.04-7.98 (m, J = 5.3, 2H), 7.79 (m, 1H), 4.35 (q, J = 7.1, 2H), 1.36 (t, J = 7.1, 3H).

13

C NMR (101 MHz, DMSO-d6) δ 165.9,

158.7, 155.5, 152.0, 150.2, 144.5, 143.8, 134.7, 130.4, 128.9, 128.6, 125.3, 124.7, 124.2, 122.1, 115.2, 61.0, 14.7. IR (KBr) ν 3418, 2990, 2565, 1730, 1632, 1579, 1510, 1442, 1380, 1295 cm-1. HRMS: calcd. for C22H18O2N4Cl+ [M+H+] 405.1124; found 405.1104. N-(4-(Methylsulfonyl)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (34). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.5 g, 2.07 mmol), 28 ACS Paragon Plus Environment


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4-(methylsulfonyl)-aniline (0.35 g, 2.07 mmol) and HCl (0.5 mL of 4M solution in dioxane) according to the general procedure A. Recrystallization from MeOH yielded 0.60 g (70%) of the title compound as a colourless solid. Salt: mp 210-213 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.69 (bs, 1H), 9.04 (d, J = 6.6 Hz, 2H), 8.84 (d, J = 8.4 Hz, 1H), 8.77 (d, J = 6.6 Hz, 2H), 8.32 (d, J = 8.8 Hz, 2H), 8.04 (m, 4H), 7.81 (ddd, J = 8.3, 5.1, 3.1 Hz, 1H), 3.27 (s, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 158.2, 155.1, 151.1, 149.8, 144.4, 143.7, 135.1,

134.3, 128.5, 128.2, 127.9, 124.1, 123.7, 121.9, 114.7, 43.9. IR (KBr) ν 3425, 2611, 1634, 1597, 1566, 1512, 1414, 1293, 1146, 952 cm-1. HR-MS (ESI) [M+H+] calcd. for C20H17O2N4S+ [M+H+] 377.1067, found 377.1065. N-(4-(Methylsulfinyl)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (35). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.28 g, 1.16 mmol), 4(methylsulfinyl)benzenamine (0.18 g, 1.16 mmol) and HCl (0.29 mL of 4M solution in dioxane) according to the general procedure B. Column chromatography (10/1 DCM/MeOH) yielded 0.19 g (45%) of the free base that was converted to the corresponding hydrochloride salt by treatment with HCl. Base: 1H NMR (400 MHz, DMSO-d6) δ 10.19 (bs, 1H), 8.78 (dd, J = 4.5, 1.5 Hz, 2H), 8.65 (d, J = 8.4 Hz, 1H), 8.31 (dd, J = 4.5, 1.6 Hz, 2H), 8.28-8.12 (m, 2H), 7.96 (dd, J = 4.5, 1.1 Hz, 2H), 7.88-7.77 (m, 2H), 7.73 (ddd, J = 8.3, 4.9, 3.4 Hz, 1H), 2.82 (s, 3H). Salt: mp 249-251 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 10.50 (bs, 1H), 9.00 (d, J = 6.5 Hz, 2H), 8.78 (d, J = 8.3 Hz, 1H), 8.69 (d, J = 6.5 Hz, 2H), 8.22 (d, J = 8.7 Hz, 2H), 8.12-7.96 (m, 2H), 7.87-7.73 (m, 3H), 2.82 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.7, 156.0, 150.6, 150.2, 146.0, 141.6, 141.2, 134.6, 128.8, 128.3, 124.8, 124.1, 123.9, 123.1, 115.1, 43.7. IR (KBr) ν 3433, 2539, 1634, 1597, 1510, 1416, 1377, 1330, 1030, 840 cm-1. HR-MS (ESI) [M+H+] calcd. for C20H17ON4S+ [M+H+] 361.1118, found 361.1116. N-(4-(Methylthio)phenyl)-2-(pyridin-4-yl)quinazolin-4-amine hydrochloride (36). The title compound was prepared from 4-chloro-2-(pyridin-4-yl)quinazoline 9 (0.50 g, 2.07 mmol), 4aminothioanisole (0.29 g, 2.08 mmol) without any HCl catalysis according to the general procedure B. Column chromatography on silica gel (10/1 DCM/MeOH) furnished 0.52 g (72%) of the free base that was converted to the corresponding hydrochloride salt by treatment with HCl. Base: 1H NMR (400 MHz, DMSO-d6) δ 10.01 (bs, 1H), 8.76 (dd, J = 4.5, 1.6 Hz, 2H), 8.61 (d, J = 8.4 Hz, 1H), 8.29 (dd, J = 4.5, 1.6 Hz, 2H), 7.94 (m, 4H), 7.69 (ddd, J = 8.3, 4.7, 3.5 Hz, 1H), 7.55-7.32 (m, 2H), 2.54 (s, 3H). Salt: mp 183-185 °C (MeOH). 1H NMR (400 MHz, DMSO-d6) δ 0.51 (bs, 1H), 9.01 (d, J = 6.6 Hz, 2H), 8.78 (d, J = 8.3 Hz, 1H), 8.68 (d, J = 6.6 Hz, 2H), 8.11-7.93 (m, 2H), 7.94-7.81 (m, 2H), 7.74 (ddd, J = 8.2, 6.7, 29 ACS Paragon Plus Environment

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1.6 Hz, 1H), 7.50-7.23 (m, 2H), 2.54 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.7, 155.2, 151.7, 148.9, 144.0, 136.2, 134.5, 133.9, 128.4, 127.9, 126.9, 124.7, 124.2, 124.0, 114.9, 15.8. IR (KBr) ν 3428, 1633, 1593, 1512, 1495, 1417, 1371, 1327, 1230, 793 cm-1. HR-MS (ESI) [M+H+] calcd. for C20H17N4S+ [M+H+] 345.1168, found 345.1167. AlphaScreen assay The AlphaScreen assay is based on energy transfer between two types of beads. Upon illumination by laser at 680 nm, donor beads containing the photosensitizer phthalocyanine convert ambient oxygen to an excited form of singlet oxygen 1O2 which can, within its halflife of 4 µs, diffuse approximately 200 nm. If an acceptor bead is within that proximity, energy is transferred from the singlet oxygen to thioxene derivates within the acceptor and a signal at 520 - 620 nm is emitted. All AlphaScreen experiments were performed in white 384-well ProxiPlates (PerkinElmer, Boston, MA, USA) using phosphate buffered saline (PBS) supplemented with 0.05% Tween 20 and 2 mM dithiothreitol (DTT). Streptavidin-coated donor beads (Perkin Elmer) were used to capture the CAI-biotin peptide. The GST fusion proteins in the binding assay were captured by Acceptor beads coated with glutathione (GSH, custom product, PerkinElmer). Equal concentration of donor and acceptor beads (5-10 µg/ml depending on the batch) were used. In the counterscreen, biotinylated GST (bGST) was used instead of CAGST and CAI-biotin in order to detect compounds disturbing interactions other than CA:CAI. Signals were detected by an EnVision plate reader (PerkinElmer). In this study 2 libraries were screened: DIVERSet library from ChemBridge (20,000 compounds; ChemBridge Corporation, San Diego, USA); LeadQuest Tripos Library (50,000 compounds (Tripos Discovery Research Centrem Bude, UK) available at Chemical Core Biology Facility of EMBL, Heidelberg, Germany. The DIVERSet library from ChemBridge (20,000 compounds; ChemBridge Corporation, San Diego, USA) was transferred from original source 96-well plates (80 compounds/plate;

250

plates)

to

384-well

polypropylene

V-bottom

plates

(320

compounds/plate; 63 plates; Greiner, Frickenhausen, Germany), diluting compounds from 10 mM in 100% dimethylsulfoxide (DMSO) on the source plate to 400 µM, 4% DMSO on the daughter plate using an Evolution P3 pipetting robot (PerkinElmer). Aliquots of this dilution were transferred to white 384-well ProxiPlates,sealed with aluminum cover foil and stored at –20 °C until use. LeadQuest Tripos library and library of natural extracts was provided already in 384-well format. 30 ACS Paragon Plus Environment


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A subset of 3,200 compounds of DiverSet Chembridge library and 3,000 compounds of LeadQuest Tripos library were tested in duplicates in a pilot screen to ascertain reproducibility. The remaining compounds were tested as singlets in lead screen. The compounds in DIVERSet and LeadQuest libraries were screened at a final concentration of 40 µM (in 0.4% DMSO, respectively 1% DMSO) for inhibitors of the CA/CAI interaction. The library of natural healing herb extracts was analyzed in both screen and counterscreen in duplicates at concentrations 10 µg/ml. A sufficient volume of 10/4.5-fold concentrated CA-GST/GSH Acceptor bead complex, and CAI-biotin/Streptavidin Donor complex was prepared and incubated for 1 h at RT in the dark. Assay plates were equilibrated at RT, centrifuged and unsealed. Thereafter, 4.5 µl of the CA-GST/GSH Acceptor bead complex and 4.5 µl of the CAI-biotin/Streptavidin Donor bead complex were added simultaneously to 1 µl of 400 µM compound in 4% DMSO using a FlexDrop dispenser (PerkinElmer) and incubated for 2 h at RT before reading the plates. The final concentrations of CA-GST, CAI-biotin and AlphaScreen beads were 60 nM, 30 nM and 10 µg/ml, respectively, in final reaction volume of 10 µl. Controls to check for maximal signal (columns 1,2; 0.4% or 1% DMSO instead of compounds, otherwise all components of the assay left unchanged as described aboveor background signal (column 23,24; PBS, 0.05% Tween 20, 2 mM DTT, 1% DMSO) were included on each plate. The data were evaluated using ActivityBase software (IDBS, Guildford, UK), and the inhibition values for all compounds

were

calculated

based

on

this

formula: Inhibition [%] =

(signal well - mean of the min. signal control) x 100 (mean of the max. signal control - mean of the min.signal control)

Z’ factor as a parameter for the quality of the assay itself was calculated using the following formula31: Z‘ =1 -

(

3 X (standard deviation of the maximum signal control) + 3 X (standard deviation of the minimum signal control) |(mean of the maximum signal control - mean of the minimum signal control)|

)

The coefficient of variation (CV) for each plate was calculated according to the following equation:

, where σ and µ represent the standard deviation and the mean of a

population of values, respectively.


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Nephelometry Solubility of compounds was measured by nephelometry using a NEPHELOstar Microplate reader (BMG). The plates for nephelometry were generated in parallel with the plates for IC50 determination. 4 µl of compound at various concentrations (serial dilutions 2 µM – 2 mM in in 20% DMSO) were transferred to 384-well clear bottom plates (Costar, #3711) and 36 µl PBS were added directly before the solubility was measured. Expression and purification of recombinant CA Coding sequences for full-length CA with N-terminal glutathione-S-transferase (GST) fusion, full-length CA (1-231) and C-terminal domain (residues 146-231) of CA were cloned into the pET11c expression vector using NdeI/BamHI sites. The CA coding region was amplified by PCR from viral genome based on NL4-3 (Adachi). The mutations were introduced using PCR and the set of oligonucleotides containing required substitutions. The recombinant CA proteins were expressed in E.coli BL21(DE3)RIL (Novagen) as soluble proteins. Bacteria were harvested and lysed by homogenization in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA by EmulsiFlex C-3 (Avestin). The CA proteins were enriched from the bacterial lysate by fractionated ammonium sulphate precipitation (20% 50%saturation). The precipitate was dissolved in 50 mM sodium acetate, pH 5.5, 10% glycerol, 2 mM 2-mercaptoethanol and dialyzed against this buffer overnight at 4 °C. The material was loaded onto a MonoS 10/100 GL cation-exchange column (GE Healthcare Life Sciences) and bound protein was eluted with a linear gradient from 0 to 800 mM NaCl in 50 mM sodium acetate, pH 5.5, 10% glycerol, 2 mM 2-mercaptoethanol. Fractions containing the proteins of interest were pooled, dialyzed against 50 mM sodium acetate, pH 5.5, 10% glycerol, 2 mM 2-mercaptoethanol, 150 mM NaCl and proteins were finally purified by size exclusion chromatography using a Superdex75 HR 10/30 prep grade column (GE Healthcare Life Sciences). The full-length CA (1-231) based on NL4-3 (Adachi) was cloned into the pGEX expression vector (GE Healthcare Life Sciences). CA with N-terminal GST fusion was purified from cleared bacterial lysate by affinity chromatography using GlutathioneSepharose 4 Fast Flow (GE Healthcare Life Sciences). Protein was bound to the column in phosphate buffered saline (PBS) and eluted with 50 mM Tris-HCl pH 8.0, 10 mM reduced glutathione. Final purification was performed on a Superdex 75 HR 10/30 column using PBS, 2 mM DTT as a running buffer. The protein solution was supplemented with glycerol (50% v/v) and stored at -20 °C.



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For high-throughput screening, the CAI peptide was extended by a 5 amino acid C-terminal linker (GGGSC). Iodoacetyl-LC-Biotin with a spacer arm of ~27Å was covalently coupled to the cysteine residue in the linker peptide. The labeled peptide inhibited in vitro assembly of Gag derived proteins with the same efficiency as CAI. The control peptide (CAIctrl) displayed the identical amino acid composition as CAI but with a scrambled sequence (IYDPTLYGLEFD). All peptides were obtained as lyophilized salts of trifluoroacetic acid (Peptide Specialty Laboratory, Heidelberg) and dissolved in 100% DMSO at a concentration of 10-20 mM). Isothermal titration calorimetry The binding characteristics of CAI peptide were determined using an Auto-iTC200 titration microcalorimeter (MicroCal Inc./Malvern Instruments Ltd., UK) at 25 °C. The reactants were prepared in 30 mM MES buffer, pH 6.0 containing 10% glycerol, 2mM 2mercaptoethanol and 2% DMSO. The exact concentrations of CA proteins as well as CAI peptide were determined by HPLC amino acid analysis. CAI peptide at concentration 320 µM – 350 µM was injected in 2 µl increments into the sample cell containing 200 µl of 23 µM – 33 µM CA or CA CTD proteins until complete saturation. Each titration was accompanied by the corresponding control experiment in which CAI peptide was injected into the buffer alone. To estimate whether binding is accompanied by proton transfer, titrations in two buffers with different ionization enthalpies were performed. The binding of the CA ligand 33 to the HIV capsid protein and its C-terminal domain was monitored using a VP-ITC microcalorimeter (MicroCal Inc./Malvern Instruments Ltd., UK) at 25 °C. The solutions of reactants were prepared in 30mM MES, pH 6.0 containing 10% glycerol, 2mM 2-mercaptoethanol, 2% DMSO, and the exact concentrations of proteins were determined by HPLC amino acid analysis. Due to low solubility of compound 33 and observed precipitation at concentrations required for ITC measurement, the ligand concentration in the prepared solution was estimated by UV spectral analysis after thorough centrifugation, and the degree of precipitation was estimated by comparison of spectra of precipitates diluted in DMSO. Typically, 9 µl aliquots of 31 µM 33 were injected stepwise into the sample cell containing 1.43 ml of 3.9 µM CA dimer or 3.1 µM CA CTD dimer until saturation was achieved. The experiment was accompanied by a corresponding control experiment in which ligand was injected into the buffer alone. The thermodynamic parameters were determined by MicroCal software implemented in Origin 7.0 (MicroCal Inc./Malvern Instruments Ltd., UK).


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Inhibition of HIV-1 replication MT-4 T-cells were grown in RPMI-40 medium (Invitrogen). HeLa TZM-bl indicator 32

cells were kept in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen). Media were supplemented with 10% fetal bovine serum (FBS) (Biochrom), 100 units/ml penicillin, 100 µg/ml streptomycin (Pen/Strep; Biochrom), and 10 mM HEPES. Virus stocks of HIV-1 NL4-333 for infectivity assays were obtained by co-culture of infected and uninfected MT-4 cells as described earlier.34 Tissue culture supernatant was harvested, cleared by filtration (0.45 µm filter) and stored at -80 °C until use. TZM-bl cells were seeded in 96-well plates and infected on the following day with virus stock at a multiplicity of infection of 1. At 6 h post infection (p.i.), tissue culture supernatants were removed and replaced by fresh complete DMEM supplemented with various concentrations of compounds. At 30 h p.i., supernatants were removed and replaced by fresh medium containing the same concentration of the respective compound. At 52 h p.i., supernatants were harvested and stored at -20 °C until use. Infectious virus released from the cells was quantitated by titration of supernatants on fresh TZM-bl indicator cells in 96-well plates. At 48 h p.i., infected cells were quantitated using a standard luciferase based assay (SteadyGlo, Promega), according to the manufacturer’s instructions. Initial screening was performed in triplicate at a final concentration of 10 µM. Compounds which showed antiviral activity at this concentration and were not found to be cytotoxic were further characterized by titration experiments from 0.625 to 20 µM. IC50 values were determined by analysis of luciferase activity per µl of tissue culture supernatant using GraphPad Prism. Cell viability measurements Cytotoxicity was determined by titration of compounds on TZM-bl cells, followed by incubation at 37 °C, 5% CO2 for 48h. Cell viability was then measured using a standard MTS cell proliferation assay (CellTiter 96 AQueous One; Promega) following the manufacturer’s instructions. Initial screening was performed in triplicate at a final concentration of 10 µM. Compounds which were not found to be cytotoxic at this concentration and showed antiviral activity were further characterized by titration experiments from 0.625 to 20 µM. Values were normalized to the value obtained for cells treated with DMSO alone.



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ASSOCIATED CONTENT Supporting Information : Procedures used for preparation of non-commercially available anilines Synthesis of quinazolines General procedures for the synthesis of substituted quinazoline-4-amines Competition of CAI-Biotin with soluble free CAI or scrambled control peptide Assay validation Antiviral assay and cytotoxicity determination Copies of 1H and 13C spectra

Corresponding Author Information : Dr. Jan Konvalinka, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences and IOCB Research Center, Flemingovo n. 2, 166 10 Prague 6, Czech Republic Fax : +420 220 183 578; Tel : +420 220 183; e-mail : [email protected]

Prof. Dr. Hans-Georg Kraeusslich, Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 691 20 Heidelberg, Germany Fax: +-49-6221-56-5003; Tel: +49-6221-56-5001; e-mail : [email protected]

Acknowledgements The authors would like to thank Iva Flaisigová for expert technical help and Hillary E. Hoffman for manuscript editing. The financial support of the grant 13-19561S from the Grant Agency of the Czech Republic and 7th Framework Program of the EU (FP7-HEALTH201095) is gratefully acknowledged. We thank the Chemical Biology Core Facility (CBCF) at the European Molecular Biology Laboratory (EMBL) and its partners, the German Cancer Research Centre (DKFZ) and University of Heidelberg, for support.

Abbreviations used : CA, capsid; CAI, capsid assembly inhibitor; CTD, C-terminal domain; GST, glutathione Stransferase; HIV, human immunodeficiency virus; ITC, isothermal titration calorimetry; MA, matrix; NC, nucleocapsid; NTD, N-terminal domain; PR, protease; SP, spacer peptide


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Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal

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