WO2020263164A1 - Compounds targeting dual g-quadruplex dna and stat3 - Google Patents

Abstract

The present invention relates to novel quinazoline compounds having the formula (I) or (II): (I) (II). The compounds are active both as stabilizers of G-quadruplex DNA structures and as inhibitors of STAT3 phosphorylation. The disclosed compounds are useful in medical treatment, such as the treatment of cancer.

Description

COMPOUNDS TARGETING DUAL G-QUADRUPLEX DNA AND STAT3

TECHNICAL FIELD

The present invention relates to novel quinazoline compounds which are active both as stabilizers of G-quadruplex (G4) DNA structures and as inhibitors of STAT3 phosphorylation. The disclosed compounds are useful in medical treatment, such as the treatment of cancer and viral infections.

BACKGROUND ART

G-quadruplex (G4) DNA structures are four-stranded secondary DNA structures that play important roles in regulating gene expression. In the human genome, it is estimated that G4 structures can form at over 700,000 positions, and they are over-represented in oncogenes and regulatory genes and are under-represented in housekeeping and tumor suppressor genes (Eddy and Maizels, 2006; Huppert and Balasubramanian, 2007). G4 structures are thus suggested to be promising chemotherapeutic targets. This is further supported by the high occurrence of G4 structures in the telomeres and by their ability to obstruct DNA replication and repair, which leads to activation of the DNA damage response pathway resulting in apoptosis of cancer cells (Shay and Bacchetti, 1997). Furthermore, cancer cells possess more G4 DNA structures compared to non-cancerous cells (Biffi et al., 2014), and clinical trials have been conducted with G4-stabilizing compounds for treatment of BRCAl/2-deficient tumors (Xu et al., 2017) and carcinoid and neuroendocrine tumors (Dry gin et al., 2009).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays important roles in cell growth and survival. Activation of the members of the STAT family of proteins through phosphorylation is thus tightly regulated, and the loss of this control is correlated with pathological conditions. In particular,

uncontrolled/constitutive active STAT3 is frequently detected in several cancer types, such as breast cancer, lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, melanoma, leukaemias, and lymphomas (Al Zaid Siddiquee and Turkson, 2008; Kamran et al., 2013; Sansone and Bromberg, 2012) and STAT3 is therefore considered to be a promising cancer drug target (Yu et al., 2014). Unphosphorylated and inactive STAT3 exists in a monomeric state and localizes mainly in the cytoplasm. When STAT3 is phosphorylated, it dimerizes and translocates into the nucleus where it promotes transcription of target genes, many of which are oncogenes (Wong et al., 2017). Thus, inhibition of STAT3 phosphorylation blocks its activation and represents one of the main strategies in STAT3-related drug development (Fagard et al., 2013).

Jamroskovic et al. (2016) screened more than 30,000 compounds for their ability to bind to three different G4 structures, and identified a quinazoline-based compound (shown as compound 5b in Figure 6) that is able to bind and stabilize G4 structures.

LaPorte et al. (2014) (LaPorte et al., 2014) disclose a similar quinazoline compound that has been shown to inhibit the STAT3 pathway in tumor cells and to act as an adenosine- receptor antagonist.

WO 2005/030131 and US 2005/0124562 (Replidyne, Inc.) disclose bis-quinazoline compounds based on the compound (3,4-dihydro-quinazolin-2-yl)-quinazolin-2-yl- amine, and methods of use of the compounds in the treatment of bacterial infections. However, there is no disclosure of compounds active as stabilizers of G-quadruplex DNA structures and/or as inhibitors of STAT3 phosphorylation.

Consequently, there is a need for new chemical compounds that are active both as stabilizers of G-quadruplex (G4) DNA structures and as inhibitors of STAT3 phosphorylation. It is predicted that such compounds are useful in medical treatment, such as the treatment of cancer.

DESCRIPTION OF THE DRAWINGS

Figure 1. Compounds 4f and 8g selectively stabilize G4 structures in vitro.

(a) Chemical structures of 4f and 8g. Dose response analyses of the Taq-polymerase stop assay with (b) 4f and (c) 8g with the different G4 templates (hybrid tel om eric G4: , parallel ribosomal and c-MYC Pu24T G4, and antiparallel cdcl3⁺ promoter DNA) and non-G4 DNA templates used in the primary Taq-polymerase assay screens. Estimated IC50 values with 4f was 4 mM for ribosomal parallel G4, 3 mM for hybrid G4 structure, and 0.3 mM for c-MYC Pu24T parallel G4 structure For 8g, the estimated IC50 for antiparallel G4 was 34 mM; ribosomal parallel G4 3 mM, and hybrid G4 17 mM, 6 mM for c-MYC Pu24T parallel G4 DNA. Binding of 4f to the c-MYC Pu24T G4 DNA structure was measured by SPR showing (d) the sensorgrams and (e) the dose-response curve as well as by MST analysis showing (f) the binding curves and (g) the dose- response curve. Concentrations of the compounds used in SPR were 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 mM and MST were 1250, 625, 312.5, 156, 78, 39, 19.5, 9.8, 4.9, 2.4, 1.2, 0.6, 0.3, and 0.15 nM. (h) Cartoon showing the structure of c-MYC Pu24T G4 DNA and the interactions of the different guanines in the presence of 4f based on the NMR data (cf. Figures 9a and b). (i) Side and (j) top views of one of the largest MD clusters of the c-MYC Pu24T G4 DNA-4f complex (k) 4f forms hydrogen bonds (dashed lines) with the amine in G-8. The observed chemical shift changes in the NMR data are shown as transparent (large shifts), dark grey (moderate shifts), and light grey (no/small shifts).

Figure 2. HeLa cells are sensitive to the novel compounds resulting in replication stress, DNA damage, and apoptosis. Resazurin-based cell viability assay of (a) HeLa and (b) HPFs treated for 48 h with 5b, 4f, or 8g at the indicated concentrations. Data represent the mean ± SD, n > 3. (c) Schematic of the DNA fiber analysis (d)

Representative images of replication tracts with different lengths. Intact DNA fibers displaying iodo-deoxyuridine (IdU) labels (light grey) flanked by chloro-deoxyuridine (CldU) labels (dark grey) (e) Quantification of the fiber length (kb) in treated (8g) versus mock cells (-). Data represent populations of individual DNA fibers for each condition of the final experiment (63 for control and 52 for treatment). The mean ± 2SD is indicated. Welch-corrected two-sample t-tests of ln-transformed data were used, and the p-value is indicated (f) Immunoblot analysis of soluble (for pATM, ATM, and PCNA) and chromatin-bound (for gH2A.C and H2AX) protein fractions extracted from HeLa cells treated for 12 h with 5b, 4f, or 8g at the indicated concentrations (g) Quantification of the immunoblot analysis in (d). Error bars represent the mean ± absolute error (n = 2) for 5b and 4f and the mean ± SD (n = 3) for 8g. (h) The number of apoptotic cells (annexin V-positive cells) measured by flow cytometry. HeLa cells were treated for 12 h with 8g at the indicated concentrations and stained with propidium iodide and annexin V. Data represent the mean ± SD, n = 3. Analysis of the data was performed using two-sample t-tests with assumed equal variance, and p-values are indicated.

Figure 3. Treatment with 4f and 8g results in increased BG4 foci in HeLa cells.

(a) Representative images of HeLa cells stained with the BG4 antibody after treatment for 12 h. (b and c) Quantification of BG4-positive cell nuclei. Data represent populations of individual cells for each condition of the final experiment (b: DMSO (-)

= 133 cells, 8g 20 mM = 111 cells; c: DMSO (-) = 130 cells, 4f 50 mM = 85 cells), and means ± 2SD are indicated. Analysis of the data was performed using Welch-corrected two-sample t-tests of ln-transformed data, and p-values are indicated.

Figure 4. Phosphorylation of STAT3 is inhibited by direct binding of 4f and 8g to the STAT3 protein, (a) Total cell lysate from HeLa cells treated for 12 h at the indicated concentrations of 5b, 4f, or 8g immunoblotted with the indicated antibodies. Actin was used as the loading control (b) Quantification of the immunoblot analysis in (a). Error bars represent the mean ± absolute error of two independent experiments. SPR sensorgrams of (c) 4f and (d) 8g binding to STAT3 protein with corresponding (e) dose response curves (the TFA salt of 8g was used to increase the solubility of 8g within the concentration range used in this experiment). The average of three measurements is shown, and full graphs are presented in Figure 14.

Figure 5. 8g localizes into nucleus in S. pombe cells and prolongs cell growth during S-phase. (a) Representative image of S. pombe cells stained by 25 mM 8g and 0.25 % DMSO (-). (b) The number of doublings per 12 hours of S. pombe cells treated with 8g. Error bars represent ±SD, n = 3. (c) Representative images of S. pombe cells from (b) treated with 0.25% DMSO (-) or 10 mM 8g. Arrows indicate“pear” -like and other cell morphology deformations (d) FACS analysis of synchronized S. pombe cells grown in PMG media treated with 5 mM and 10 mM of 8g. Representative FACS profiles are shown, n = 3. (e) Quantification of the fiber length (kb) in treated (8g) versus mock cells (-). Data represent populations of individual DNA fibers for each condition of the final experiment (n=40 per treatment). The mean ± SD is indicated.

Two-sample t-tests was used, and the p-value is indicated.

Figure 6. (a) Synthesis of quinazoline-pyrimidine, quinazoline-quinazolinone and pyrimidine-pyrimidine derivatives; (b) Tautomeric forms of compounds 8 (a-g), 9 (a-f) and 12-17.

Figure 7. Primary screening of the synthesized compounds in a Taq-polymerase stop assay using S. pombe with (a) parallel ribosomal G4 DNA, (b) hybrid telomeric G4 DNA, (c) antiparallel cdcl3⁺ promoter G4 DNA, and (d) nonG4 DNA as a control. All graphs represent the mean of two independent experiments ± absolute error. Arrows indicate the compounds selected for further study (f-g) Another set of synthesized compounds screened with the Taq-polymerase stop assay. Normalized full-length product of synthesis of (f) parallel c-MYC Pu24T G4 DNA and (g) non-G4 DNA. All compounds in f-g selectively inhibit DNA synthesis on non-G4 DNA.

Figure 8. (a) Dose response analyses of Taq-polymerase stop assay of 5b with the different G4 DNA and non-G4 DNA templates used in the primary screen. The estimated IC o for ribosomal parallel G4 17 mM, hybrid G4 ~30 mM, and 3 mM for c- MYC Pu24T parallel G4 DNA. SPR sensorgrams demonstrating the binding of 4f to (b) S. pombe parallel ribosomal G4 DNA, (c) human parallel c-kit G4 DNA, (d) S. pombe hybrid telomeric G4 DNA, and (e) ssDNA. SPR sensorgrams demonstrating the binding of 8g to (f) human c-myc Pu24T G4 DNA, (g) S. pombe parallel ribosomal G4 DNA,

(h) human parallel c-kit G4 DNA, (i) S. pombe hybrid telomeric G4 DNA, and (j) ssDNA.

Figure 9. Binding mode of 4f with human c-myc Pu24T G4 DNA. (a) The imino-region of the ¾ NMR spectrum of c-myc Pu24T in the absence (bottom) and presence of 0.5 equivalents 4f (middle) and 1 eq 4f (top). A new set of well-defined peaks appeared upon addition of 4f, originating from the 4f:DNA complex. At a 1 :2 ratio of 4f to DNA, both free and bound forms of the imino peaks were observed, which were used to assign the bound form. Peaks marked with asterisks originated from the DNA in complex with 4f. The sizes of the induced chemical shift changes could be determined by observing exchange peaks in a 2D NOESY spectrum (b) of 0.5 eq 4f binding to c-myc Pu24T G4 DNA. Off-diagonal peaks represent cross-peaks from the exchange between free and bound form of DNA. (c) Side view and (d) top view of the chemical shift differences upon 4f binding mapped on the top of c-myc Pu24T G4 DNA. The observed chemical shift changes are divided into three intervals: >0.2 ppm (transparent), 0.1-0.2 ppm (dark grey), and <0.1 ppm (light grey).

Figure 10. (a) Emission and excitation spectra of 5 mM 8g dissolved in 100% DMSO. (b) 2-photon excitation microscopy imaging of living HeLa cells treated with 50 pM 8g for 10 and 30 min. Graphs showing the resazurin-based cell viability assay with HeLa cells or HFPs treated for 48 h with the compounds (c) 5b, (d) 4f, and (e) 8g at the indicated concentrations. Data are from the same graphs shown in figures 2a and 2b.

Figure 11. (a) Density plots showing the flow cytometry analysis of HeLa cells stained with propidium iodide and annexin V. Cells were treated for 12 h with 8g at the indicated concentrations. For each treatment, the percentages of living cells (bottom left quarter), cells in early apoptosis (bottom right quarter), cells in late apoptosis (upper right quarter), and necrotic cells (upper left quarter) are indicated (b) Representative images of HeLa cells treated for 12 h with 50 pM 4f (right) or DMSO (left). Cell nuclei were stained with DAPI upon cell fixation. Black arrows indicate ICBs. (c) Fold change in the number of ICBs in treated versus untreated cells. At least 350 cells from six technical replicates were counted for each treatment, and the data represent the mean ± SD of three independent experiments. Analysis of the data was performed using the two sample t-test. (d) The numbers of BG4-positive dots per cell after 1 h treatment with 8g at the indicated concentrations. Data represent populations of individual cells for each condition of the final experiment, and means ± 2SD are shown. The following numbers of cells were analyzed for each treatment: DMSO only: 119 cells, 8g 5 pM: 104 cells, and 8g 20 pM: 108 cells. Analysis of the data was performed using Welch-corrected two-samples t-tests. The pilot experiment, sample size determination, and effect size are reported in the source data (f) Viability of different cell lines. Cells were treated for 48 h with 7 pM 4f. Data represent the mean ± SEM (n = 3). Analysis of the data was performed using the two-sample t-test. Figure 12. Schematic overview of the S. pombe cell cycle (outer solid line circle) with the corresponding amount of DNA (C) detected in the flow cytometry analysis (inner dotted circle).

Figure 13. Model of action of 4f/8g in cancer cells.

Figure 14. SPR measurements of binding of (a) 4f, (b) 8g, and (c) 8g-TFA to STAT3 protein. Each experiment was run in triplicate. Concentrations are specified in the section“Experimental methods”.

Figure 15. Titration (a) UV/vis spectral changes of 10 mM 8g in absence (black line) and presence (gray line) of equimolar concentration c-MYC Pu24T G4 DNA. Arrow indicates isosbestic point (b-f) Fluorescence titration analysis of 2 pM 8g with different DNA and RNA molecules. 8g was excited at l=305. Titration spectra of representative experiments are shown. Error represents the fitting error of the data. Each binding curve was fitted into data from at least two independent experiments. Data of ssDNA were not fitted.

Figure 16. STAT3 SPR screen performed with 5 pM of different compounds. Error bars represent standard deviation estimated from multiple 8g-TFA measurements.

Figure 17. Determination of toxicity in HeLa cells. HeLa cells treated for 48 h with compound A) KC45 (8g), KC240 (8h), KC246 (8j), KC235 (8k); B) KC45 (8g),

KC261 (8n), KC281 (8o) and KC253 (8m); C) KC45 (8g), KC298 (8q), KC250 (81) and KC234 (8i) at the indicated concentrations. Data present mean, n>3.

Figure 18. Growth inhibition (GI) defined as logio GEo. KC45 (8g) tested in 17 different cancer tissues show that Ovary, Skin, Kidney, Myeloma and Breast cancer types are the top 5 tissue types that are most sensitive to KC45 treatment. The cells were treated for 72h.

Figure 19. BHK cell viability at various concentrations of KC45 (8g). Black: at 17h. Grey: at 44h. Figure 20. Effect of KC45 (8g) on the replication of CHIKV in BHK cells 8h post infection. (A) DMSO, (B) CHIKV VRPs and DMSO, (C) CHIKV VRPs and 2.5 mM KC45. (D) CHIKV VRPs and 5 mM KC45.

DESCRIPTION OF THE INVENTION

It has surprisingly been found that certain quinazoline compounds bind and/or stabilize G-quadruplex structures and STAT3 in vitro and that treatment of human cells with the compounds both increases the number of G-quadruplex foci and inhibits the

phosphorylation of STAT3. Furthermore, the compounds reduce cell viability by inducing replication stress, DNA damage, and apoptosis. Importantly, treated breast cancer-derived cells showed reduced viability compared to non-cancerous cells from breast tissue. The use of a single compound that both inhibits STAT3 activation and stabilizes G-quadruplex structures is thus a potential chemotherapeutic strategy.

Evidence is provided that the novel compounds 4f and 8g not only stabilize G4 structures in vitro and in human cell culture, but also are able to selectively inhibit the STAT3-mediated pathway by directly binding to the STAT3 protein (Figure 13). To the inventors’ knowledge, this is the first study that simultaneously report on compounds that both inhibit STAT3 protein phosphorylation and stabilize G4 DNA structures.

Other compounds have been independently described as capable of stabilizing G4 structures and inhibiting STAT3 activation (Arora et al., 2008; Chen et al., 2012; Cui et al., 2012; Moraca et al., 2017; Pandey et al., 2015; Wen et al., 2017).

It is shown that the compounds according to the invention induce replication stress, telomere and genome instability, and apoptosis and might therefore be beneficial for use in cancer therapy. Furthermore, the ability to affect two targets with a single drug-like low molecular weight compound represents a chemotherapeutic concept with potential benefits such as synergism and reduced drug resistance and thus is of high therapeutic relevance.

Consequently, in a first aspect, the invention relates to a compound of the formula (I) or (II), or a pharmaceutically acceptable salt, tautomer, or ester thereof, for use in the treatment or prophylaxis of a medical condition that can be treated by stabilizing G- quadruplex (G4) DNA structures and inhibiting STAT3 phosphorylation;

wherein R¹ and R² are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; or R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form

(a) substituted or unsubstituted aryl, preferably phenyl; or

(b) a substituted or unsubstituted cyclic C4-C8 alkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The said aryl, phenyl or cyclic alkyl group may optionally be substituted with one or more substituents selected from C'-C⁶ alkyl, C'-C⁶ alkoxy, (for example methoxy), nitro, halogen, amino, carboxylate and hydroxy.

The term“halogen” refers to fluoro, chloro, bromo, or iodo.

R³ is selected from the group consisting of hydrogen, methoxy, morpholinyl, halogen, amino, diethylamino, piperidinyl, and piperazinyl substituted with 2-aminobenzoyl. R⁴, R⁵, and R⁶ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C₁-C₃ alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl.

R⁷ and R⁸ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C₁-C₃ alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; or R⁷ and R⁸ together with the ring atoms to which R⁷ and R⁸, respectively, are attached form phenyl.

Preferred compounds to be used according to the invention include compounds wherein at least one of the conditions (i), (ii) or (iii) is fulfilled:

(i) R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form (a) substituted or unsubstituted phenyl, or (b) a substituted or unsubstituted cyclic C₃-C₈ alkyl group;

(ii) R⁷ and R⁸ together with the ring atoms to which R⁷ and R⁸, respectively, are attached form phenyl;

(iii) R⁸ is phenyl and at least one of RkR⁴ is methoxy.

All isomeric forms possible (pure enantiomers, diastereomers, tautomers, racemic mixtures and unequal mixtures of two enantiomers) for the compounds having formula (I) or (II) are within the scope of the invention.

In one preferred form, the compound to be used according to the invention is a compound wherein R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form (a) substituted or unsubstituted phenyl, or (b) a substituted or unsubstituted cyclic C₄-C₈ alkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The said phenyl could e.g. be substituted with hydroxy.

In compounds wherein R¹ and R² together form phenyl or cyclic alkyl, R⁵ is preferably C₁-C₃ alkyl, and most preferably methyl. Preferred compounds wherein R¹ and R² together form phenyl or cyclic alkyl also include those wherein:

R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form substituted or unsubstituted phenyl, or a substituted or unsubstituted cyclic alkyl group selected from cyclopentyl and cyclohexyl;

R³, R⁴ and R⁷ are hydrogen;

R⁵ and R⁶ are methyl; and

R⁸ is selected from the group consisting of methyl and phenyl. Examples of preferred compounds wherein R¹ and R² together form phenyl or cyclic alkyl include:

2-((4,6-di methyl pyri mi din-2-yl)amino)-4-methylbenzo[/?]quinazolin-7-ol (referred to as 4e or KC13)

A -(4,6-di methyl pyri mi din-2-yl)-4-methyl -7, 8,9, 10-tetrahydrobenzo[/?]quinazolin-2- amine (referred to as 4f or KC29)

4-methyl-/V-(4-methyl-6-phenylpyrimidin-2-yl)-7,8,9, 10-tetrahydrobenzo[/z]quinazolin- 2-amine (referred to as 7f or KC31)

A-(4, 6-dim ethyl pyri mi din-2-yl)-4- ethyl -8, 9-dihydro-7//-cyclopenta[/_?]quinazol in-2- amine (referred to as 4i or KC243)

In another preferred form, the compound to be used according to the invention is a compound wherein R⁷ and R⁸ together form phenyl, i.e. a compound having the formula (III)

wherein R¹, R², R⁴, and R⁵ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl;

and R³ is selected from the group consisting of hydrogen, methoxy, morpholinyl, halogen, amino, diethylamino, piperidinyl, and piperazinyl substituted with 2- aminobenzoyl.

In compounds of formula (III), R⁵ is preferably C1-C3 alkyl, and most preferably methyl.

Preferred compounds of formula (III) also include those wherein:

R¹ is hydrogen or methoxy;

R² is hydrogen or morpholinyl;

R⁴ is hydrogen or methoxy; and

R⁵ is methyl. Examples of preferred compounds of formula (III) include:

2-((6-m ethoxy -4-methylquinazolin-2-yl)amino)quinazolin-4(li )-one (referred to as 8a or KC41)

2-((4-m ethyl -6-morpholinoquinazolin-2-yl)amino)quinazolin-4(l //)-one (referred to as

8g or KC45)

2-((6,7-dimethoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(li )-one (referred to as 8c or KC47)

2-((4-m ethyl -7-morpholinoquinazolin-2-yl)amino)quinazolin-4( l //)-one (referred to as 8i or KC234)

2-((4-m ethyl -6-(pi peri din- 1 -yl)quinazolin-2-yl)amino)quinazolin-4( 1 //)-one (referred to

2-((6-(diethylamino)-4-methylquinazolin-2-yl)amino)quinazolin-4(li7)-one (referred to as 8j or KC246)

2-((6-bromo-4-methylquinazolin-2-yl)amino)quinazolin-4( l //)-one (referred to as 8k or KC235)

2-((6-chl oro-4-m ethyl quinazolin-2-yl)amino)quinazolin-4( l //)-one (referred to as 81 or KC250)

2-((6-fl uoro-4-m ethyl quinazolin-2-yl)amino)quinazolin-4( l //)-one (referred to as 8m or KC253)

2-((8-m ethoxy -4-methylquinazolin-2-yl)amino)quinazolin-4(l//)-one (referred to as 8n or KC261)

2-((6-(4-(2-aminobenzoyl)piperazin-l-yl)-4-methylquinazolin-2-yl)amino)quinazolin- 4(l//)-one (referred to as 8o or KC281)

2-((6-amino-4-methylquinazolin-2-yl)amino)quinazolin-4(li )-one (referred to as 8q or KC298)

In a further preferred form, the compound to be used according to the invention is a compound wherein R⁸ is phenyl and at least one of R'-R⁴ is methoxy.

In compounds of this type, R⁵ is preferably C₁-C₃ alkyl, and most preferably methyl.

Preferred compounds wherein R⁸ is phenyl and at least one of R³-R⁴ is methoxy also include those wherein:

R¹, R², R³, and R⁴ are independently hydrogen or methoxy, provided that at least one of R¹, R², R³, and R⁴ is methoxy;

R⁵ and R⁶ are methyl; and

R⁷ is hydrogen.

Examples of preferred compounds wherein R⁸ is phenyl and at least one of R³-R⁴ is methoxy include:

7-methoxy-4-methyl-A-(4-methyl -6-phenyl pyri mi din-2-yl)quinazolin-2-amine (_ref_errecj to as 7b or KC18)

5, 6, 7-trimethoxy-4-methyl-A_f-(4-rn ethyl -6-phenyl pyri mi din-2-yl)quinazolin-2-amine

(referred t

6, 7-dimethoxy -4-methyl -N-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine (referred to as 7c or KC30)

In a preferred aspect of the invention, the said medical condition that can be treated by stabilizing G-quadruplex (G4) DNA structures and inhibiting STAT3 phosphorylation is viral infection and/or cancer, such as cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, brain cancer (malignant glioma tumor cells), pancreas cancer, colon cancer, and lung cancer.

Included in the invention is also a pharmaceutical composition comprising as the active ingredient a therapeutically effective amount of a compound of formula (I), (II) or (III) as defined above, in association with at least one pharmaceutically acceptable excipient, carrier or diluent. The said pharmaceutical composition is useful for the treatment or prophylaxis of a medical condition that can be treated by stabilizing G-quadruplex (G4) DNA structures and inhibiting STAT3 phosphorylation. Examples of such medical conditions include viral infections and/or cancer, such as cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, brain cancer (malignant glioma tumor cells), pancreas cancer, colon cancer, and lung cancer.

A further aspect of the invention is a compound having the formula (III) for use as a fluorescent probe in an in vitro diagnostic method

wherein R¹, R², R³, R⁴, and R⁵ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; and provided that at least one of R² and R³ is selected from the group consisting of morpholinyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, and optionally substituted pyrrolidinyl.

EXPERIMENTAL METHODS

Statistical analysis

The minimal sample sizes for the microscopy experiments (BG4 immunostaining of human cells and S. pombe and fiber analysis) were determined by pilot experiments. Distribution plots and quantile-quantile plots were used to graphically examine the normality of the sample distributions. Transformation to natural logarithms was performed if required. P-values were calculated by two-sided Welch-corrected t-test in case of unequal variance. Einequal variance was determined by the F-test. Effect sizes and the means with asymmetric ± 2SD were calculated. In the ICB experiment and flow cytometry, a two-sided Student’s t-test with assumed equal variance was used to determine significant differences. A p-value < 0.05 was considered significant. All calculations were performed in Microsoft Excel and OriginLab software. Microscopy of BG4 immunostaining of human cells and S. pombe spheroplasts was single blinded using the DAPI channel for sample acquisition. Fiber analysis and ICB experiments were not blinded. Taq DNA polymerase assay

All DNA molecules used in the assay were purchased from Eurofms Genomic (Table 2), and the experiment was performed as described previously (Jamroskovic et al., 2016). In brief, each reaction contained 40 nM template DNA incubated with 25 mM compound, and the control reaction used 5% DMSO in place of the compound. Each reaction was run for 10 min. The final quantification in figure 7a-d was the average value of two independent experiments along with the absolute error. For the dose response analysis, 40 nM template DNA was incubated with 0.06, 0.16, 0.4, 1, 2.6, 6.4, 16, or 40 pM of the compounds or with 5% DMSO as a control reaction. The experiments were performed in the same way as in the primary screening. The final quantification was the average value of three independent experiments along with the standard deviation. IC50 values were calculated by fitting the data from each experiment to the dose-response function in the Origin 8.5 software.

Surface plasmon resonance

The SPR experiment with DNA molecules was performed on a ProteOn XPR (Biorad) at 25°C. A final concentration of 5 pM biotin-labeled oligonucleotides (Table 2) were folded into G4 structures in SPR buffer without DMSO (10 mM potassium phosphate buffer, pH = 7, 150 mM KC1, 0.05% Tween, and 5% DMSO) at 95°C for 5 min and cooled down to room temperature overnight. Folded oligonucleotides were immobilized on a neutravi din-coated NLC sensor chip (Biorad) at a rate of 30 pl/min until maximal response unit (RU) values were reached (ribosomal G4 DNA: 780 RU, telomeric G4 DNA: 720 RU, c-MYC pu24T G4 DNA: 1,020 RU, c-kit G4 DNA: 780 RU, ssDNA: 1,150 RU). Compounds 4f and 8g were injected at a flow rate of 50 pl/min for 120 s. Signal from a reference surface was subtracted, and the data were solvent corrected for DMSO in order to obtain the true RU values. The apparent binding constants (K_D) were calculated by fitting the data to a single-site binding function in the OriginLab 2016 software. All data were smoothed for visualization purposes only.

The SPR experiment with STAT3 protein was performed on a Biacore T200 (GE Healthcare). A total of 10 ng/pl of his-STAT3 protein (SignalChem) (diluted in U phosphate-buffered saline (PBS), 0.005% Tween, and 5% DMSO) was immobilized on the NTA sensor chip at a flow rate of 5 pl/min until 1,500 maximal RU. Compounds 4f, 8g, and 8g-TFA were injected in triplicate at a flow rate of 50 pl/min for 120 s. Compound 8g showed signs of aggregation, so 8g-TFA with improved solubility was used instead. The signal from a reference surface was subtracted, and the KD values were calculated by fitting the averaged data from the sensorgrams to a single-site binding function in GraphPad Prism 8.0.

The SPR experiment with STAT3 protein using KC45 (8g), KC240 (8h), KC281 (8o), KC298 (8q), KC240 (8h), KC261 (8n), KC234 (8i), KC235 (8k), KC250 (81), and KC253 (8m) was performed on a Biacore 3000 (GE Healthcare). A total of 10 ng/pL of his-STAT3 protein (SignalChem) (diluted in l x phosphate-buffered saline (PBS), 0.005% Tween 20, and 5% DMSO) was covalently immobilized on the NTA sensor chip at a flow rate of 5 pL/min until 13,000 maximal RU. 5 mM compounds (diluted into PBS, 0.005% Tween 20, and 5% final concentration of DMSO) were injected in duplicates at a flow rate of 50 pL/min for 60 s. RU signal was normalized to MW of compounds. The signal from a reference surface was subtracted. All data were analyzed in Scrubber2 software.

Microscale thermophoresis

C-myc Pu24T DNA labeled with CY5 at the 5' end was folded in 10 mM potassium phosphate and 100 mM KC1 (pH 7.4) by heating at 95°C for 5 min followed by cooling to room temperature. All experiments were performed in 10 mM potassium phosphate (pH 7.4), 100 mM KC1, 0.05 % Tween 20, and 4% BSA, and the DNA concentration was held constant at 25 nM and the 4f concentration varied from 0.15 nM to 1.25 pM (fourteen 1 : 1 dilution). The samples were loaded into standard MST-grade glass capillaries, and the MST experiment was performed using a Monolith NT.l 15 (Nano Temper, Germany) with 40% LED power. Data were analyzed using the Nano Temper analysis software, and K_d was calculated by fitting the data to the Hill equation in OriginPro 8.

Nuclear magnetic resonance

The G4 DNA stock solution was prepared by folding 200 pM c-MYC Pu24T in 10 mM potassium phosphate buffer (pH = 7.4) and 35 mM KC1 by heating to 95°C and slowly cooling to room temperature overnight. An effective DNA concentration of 180 pM was obtained by adding 10% D2O. NMR samples were prepared in 3 mm NMR tubes by adding 1 equivalents 4f or 8g to the DNA stock solution. For 4g, an additional sample with 0.5 eq compound was also prepared. All spectra were recorded at 298 K on a Bruker 850 MHz Avance III HD spectrometer equipped with a 5 mm TCI cryoprobe. Excitation sculpting was used in the ID 'H experiments, and 256 scans were recorded. The 2D NOESY experiment was recorded with 32 scans, 256 ti -increments, a relaxation delay of 1.1 s, and a mixing time of 200 ms. Processing was performed with zero-filling in the indirect dimension and using 90°-shifted squared sine-bell apodization in both dimensions for the NOESY spectrum. Processing was performed in Topspin 3.5 (Bruker Biospin, Germany).

Spectroscopic measurements

2.5 mM 8g was diluted into 100 mM KC1 and 10.0 mM TRIS pH = 7.5 with and without the same equivalent of folded c-MYC pu24T G4 DNA and UV/VIS absorption spectra were recorded by T90+ UV/Vis spectrometer (PG instruments Ltd).

Spectrofluorimetric measurement

Emission and excitation spectra of 5 mM 8g prepared in 100% DMSO were recorded in a quartz cuvette with a 1 cm path length on a Jasco Spectrofluorometer FP-6500.

Fluorimetric titrations

2.0 mM 8g (in 100 mM KC1 and 10.0 mM TRIS pH = 7.5, 0.025% DMSO) was titrated by DNA or RNA oligonucleotides folded in the same buffer. Isosbestic point, kexc =

305 nm, was used for 8g excitation and fluorescence spectra (kern = 315-675 nm) were recorded by Jasco FP-6500 spectrofluorometer. DNA/RNA background fluorescence was subtracted from all data. Peak values at lah = 546 nm were fitted into hyperbolic binding function in Graphpad Prism 8.0 available at

https://www.graphpad.com/support/faq/fitting-binding-of-fluorescent-ligands/.

S. pombe growth and doubling time

The S. pombe ( bfrl::hygr , pmdlr.natr, ade6-M210, leul) strain (Kawashima et ak, 2012a) that had genes deleted in the multi-drug resistance response was the kind gift of the laboratory of Dr. Tarun Kapoor (Rockefeller University). The cells were

exponentially grown at 30°C in minimal medium EMMII (Formedium), and 1 x 10⁶ cells/ml were treated with 8g (1.6, 3.1, 6.3, 8, 10, 12.5, 25, 50, and 100 pM) or 0.25% DMSO for 12 h. The number of doublings per 12 hours was calculated. S. pombe cell synchrony and flow cytometry analysis

S. pombe ( bfrl::hygr pmdr.natr, cdc25-22 ) cells were used, and the experiment was performed as described previously (McDonald et al., 2016). Cell were treated with 5 or 10 mM 8g immediately after G2 release. Samples were taken as described in Figure 5, and the analysis was performed on a Beckman Coulter Cytomics FC500 flow cytometer. The experiment was repeated at least three times for each condition.

S. pombe fluorescence microscopy

Cells were exponentially grown at 30°C in minimal medium PMG (Formedium). 5 x 10⁶ cells/ml were treated with 8g (final concentration: 25 pM) or 0.25% DMSO (control) for 30 min, washed in PMG medium, and immobilized on poly-L-lysine coated glass slides. Localization of 8g was immediately analyzed by confocal microscope Leica SP8 FALCON using HC PL APO 63x/1.40 OIL CS2 objective, hybrid detector and Diode 405 nm laser with recorded emission between 520 nm - 620 nm. Maximum intensity projection of Z-stack images was used for visualization. To determine nuclear localization of 8g, background intensity of the fluorescence signal was decreased in both samples by identically treating the images with the ImageJ software (Schneider et al., 2012).

S. pombe DNA fiber analysis

The S. pombe strain used for the DNA fiber analysis was (hbfir 1 : :hygr pmd: :natr cdc25- 22, pfhl⁺: :ura4⁺-nmt-pfhl-GFP, leul-32: :[hENTl leul⁺], his7-366: :[hsv-tk his7⁺], ade6-M21? ade6-M210? his3-Dl? telo-his3?. Cells were grown to 10⁷ cells/ml in the presence of 1.5 pM 8g or 0.015% (v/v) DMSO at 25°C in liquid EMM2 (Formedium) media for about 12 h. Next, the cultures were diluted to 5 X 10⁶ cells/ml and the concentration of 8g was increased to 3 pM before arresting the cells in G2 -phase at 37°C for 4 h. The cells were released from G2 -phase, by shifting the temperature back to 25°C. 66 pM final concentration of bromodeoxyuridine (BrdU) was added 30 minutes after release from the G2 -phase, and cells were allowed to incorporate BrdU into their DNA for 35 min. After addition of a stop solution (250 mM EDTA, pH 8.0, 0.16% sodium azide), cells were harvested by centrifugation and resuspended in cold 70% ethanol. 200 U/mL lyticase from Arthrobacter luteus (Sigma-Aldrich) was used to digest the cell-wall prior to stretching DNA fibers on microscopic slides. BrdU incorporated into DNA was detected using rat anti BrdU clone BUI/75 (ICRI) primary antibody (ABD Serotec) and Goat anti Rat IgG Alexa Fluor 568 secondary antibody (Life technologies), while anti -DNA antibody single stranded clone 16-19 primary antibody (Sigma Aldrich) and Goat Anti Mouse IgG2a (y2a) Alexa Fluor 488 secondary antibody (Life technologies) were used to detect ssDNA. Stained DNA fibers were visualized using Axio Imager Z1 microscope (Zeiss) and images of untangled DNA fibers were taken at random from different fields. Only DNA fibers with BrdU label having intact ssDNA ends or DNA fibers with BrdU label measuring more than 70 pm were selected for analysis using Zen 2.6 blue edition (Zeiss) and ImageJ software packages. The experiments were repeated independently twice with two biological replicates.

DNA fiber analysis for HeLa cells

Asynchronous HeLa cells at 70% confluence were seeded at 1 ^c 10⁵ cells 18 h prior to the 24 h treatment with 10 pM 8g or 0.1% DMSO (control cells). Cells were pulse- labeled with 25 pM iodo-deoxyuridine (IdU) in fresh medium containing 10 pM 8g or 0.1% DMSO for 30 min. Subsequently, cells were incubated for 30 min in fresh medium containing 200 pM chloro-deoxyuridine (CIdU) and 10 pM 8g or 0.1% DMSO, followed by a 1 h incubation in fresh medium with 200 pM thymidine. Cells were then harvested and resuspended in cold PBS. DNA fiber stretching was performed as previously described (Nieminuszczy et ak, 2016). Briefly, stretched DNA fibers were immunostained with primary antibodies for IdU detection, for CIdU detection, and for ssDNA detection along with their respective Alexa Fluor antibodies. The antibodies used and their dilutions are shown in Table 3. Stained DNA fibers were visualized using an Axio Imager Z1 microscope (Zeiss), and images were captured randomly from different fields containing untangled fibers. Only fibers containing IdU labels flanked by CIdU labels with intact ssDNA ends were selected for analysis using the ZEN 2.3 (Zeiss) and ImageJ software packages. A minimum of 131 individual DNA fibers were measured for each experimental condition in two independent experiments.

Measurements were made in micrometers and converted to kilobases using a conversion factor for the length of a labeled track of 1 pm corresponding to roughly 2 kb (Bianco et ak, 2012). Cell culture and compound preparation

HeLa cells (epitheloid cervix carcinoma, purchased from Sigma-Aldrich) and HPFs from healthy adults (a kind gift from Leonardo Salviati, University of Padova, Italy) were cultured at 37°C in 7% CO2 in DMEM high glucose medium with Glutamax (Gibco) supplemented with 1 mM sodium pyruvate, penicillin-streptomycin, and 10% (for HeLa) or 20% (for HPFs) fetal bovine serum_. Cells were tested to confirm the absence of mycoplasma. Compounds were dissolved in DMSO to 10 mM (4f) or 20 mM (8g and 5b), aliquoted, and stored at -20°C. Prior to the addition to cells, the compounds were dissolved in the culture medium at the final concentration required.

Cell viability

Cell viability was measured using the PrestoBlue cell viability reagent (Invitrogen) according to the manufacturer’s recommendations. Briefly, 5,000 (for HeLa) or 4,000 (for HPFs) cells/well were seeded in complete medium on 96-well plates the day before the treatment. Compounds were dissolved in medium at the indicated concentration and added to cells. At 48 hours after treatment, 10 mΐ of PrestoBlue was added to each well and the cells were incubated at 37°C for three additional hours. Fluorescence

(Excitation 560 nm, Emission 590 nm, 10 nm bandwidth) was recorded using a Synergy H4 microplate reader (Biotek).

Protein extraction and immunoblotting

For H2A.X and ATM analysis, HeLa cells were seeded on 10 cm dishes the day before treatment in order to have 80-90% confluency the day after. Cells were treated for 12 h at the indicated concentrations. Detergent-solubilized protein fractions (for

ATM/pATM analysis) and nuclear histone-bound protein fractions (for H2A.X/yH2A.X analysis) were extracted as previously described (Li et ak, 2018). For STAT protein analysis, HeLa cells were seeded on 6-well plates the day before treatment in order to have 80-90% confluency the day after. Cells were treated for 12 h at the indicated concentrations and solubilized for 30 min on ice in RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 50 mM Tris-HCl (pH 8.0)). After high-speed centrifugation, the supernatant was collected for further analysis. Buffers for protein extraction were supplemented with 1 x EDTA-free Halt protease inhibitor cocktail (ThermoFisher Scientific), 1 mM NaOV₄, and 3 mM NaF. Protein amounts were quantified using a BCA protein assay kit (ThermoScientific). Equal amounts (15 qg) of protein were separated on 4-20% SDS-TGX (Bio-Rad) gels and transferred to 0.45 mM nitrocellulose membranes (GE Healthcare Life Sciences) using a Mini-Protean electrophoresis system (Bio-Rad). Membranes were blocked in 5% non-fat milk for 2 h. Primary antibodies were incubated overnight at 4°C, and horseradish peroxidase-conjugated-secondary antibodies were incubated 1 h at room temperature. The antibodies used and their dilutions are shown in Table 3. All washes and incubations were performed in Tris-buffered saline with Tween 20. Chemi luminescent detection was performed using ECL western blotting substrates

(ThermoScientific) and a ChemiDoc Touch Imaging System (Bio-Rad). Signal quantification was performed using the ImageQuant TL software (GE Healthcare Life Sciences).

Apoptosis assay

HeLa cells (120,000 cells/well) were seeded on 6-well plates the day before the treatment. Cells were treated for 12 h with 8g at the indicated concentrations, and the number of apoptotic cells was detected by flow cytometry using the FITC/Annexin V Dead Cell Apoptosis Kit (Molecular Probes) according to the manufacturer’s instructions. Propidium iodide/FITC Annexin V-stained cells were measured with a Cytomics FC500 (Beckman Coulter) equipped with a 488 nm argon laser. A total of 30,000 cells were collected for each sample. Propidium iodide emission was detected on the FL4 channel (675 nm), and FITC emission was detected on the FL1 channel (525 nm), and the FL4 channel was manually compensated over the FL1 channel. Data were analyzed with the CXP Analysis software (Beckman Coulter). The analysis was performed on ungated cells, quadrants were determined on the untreated sample, and the same parameters were used for analyzing all samples.

BG4 immunostaining

BG4 immunostaining was performed using a protocol modified from (Biffi et al., 2013). Briefly, 60,000 cells were seeded on 13 mm glass coverslips the day before treatment. After treatment with the compound, the cells were fixed in 2% paraformaldehyde and permeabilized in 0.1% Triton X-100 at room temperature. Cells were blocked in 2% non-fat milk followed by incubation with primary, secondary, and Alexa Fluor- conjugated antibodies. Each incubation was for 1 h at 37°C in a humidified chamber. The antibodies used and their dilutions are shown in Table 3. All washes and incubations were performed in l x PBS buffer. Cell nuclei were stained with 0.2 pg/ml diamidino-2-phenylindole (DAPI) solution prior to mounting the coverslips on glass slides with DAKO mounting medium (Agilent Technologies). Cells were imaged with a Zeiss Axiolmager Z1 equipped with an Apotome and a 63 ^c oil objective (NA 1.40) using identical acquisition settings. Cell nuclei were focused on the DAPI channel, and BG4-positive foci were counted in a semi-automatic mode using a customized Cell Profiler (Broad Institute) pipeline. All images were processed using ImageJ software.

In vivo cell microscopy

Around 100,000 cells were seeded the day before treatment on glass-bottomed microwell dishes (MaTek corporation). The cells were treated with 50 mM 8g for the indicated time points and then washed with 1 ^c PBS and resuspended in complete DMEM medium without phenol red and supplemented with 25 mM Hepes.

Fluorescence was imaged within 20 minutes from the end of compound treatment with a Scientifica 2P galvo microscope equipped with a Spectra Physics Mai Tai DeepSee Tksapphire laser. Acquisition was made with a 20x water immersion objective designed for 2-photon applications (Olympus XLUMPLFN 20XW, NA = 1). Fluorescence was detected with two GaAsP PMTs from Hamamatsu (emission filter green 525 nm/50, red 585 nm/40). The voltage was kept at 700V. For CLSM, HeLA cells were treated with 20 mM 8g for 30 min in DMEM medium. After 30 min, DMEM medium was replaced by DMEM medium without phenol red and cells were imaged by the confocal microscope Leica SP8 FALCON using HC PL APO 63x/1.40 water CS2 objective and Diode 405 nm laser with 4% power to avoid auto-fluorescence of cells. Emission was recorded between 520 nm - 620 nm by hybrid (HyD detector). Maximum intensity projection of Z-stack images was used for visualization and final images were processed using Fiji (ImageJ) software. Fluorescence signal in treated and untreated images was enhanced for visualization purpose only. For quantification, regions of interest were selected in cell cytoplasm and nucleoli, and the average fluorescence signal from the selected areas were used. ICB assay

About 60,000 HeLa cells were seeded on 13 mm glass coverslips the day before treatment. Cells were treated for 12 h with 50 mM 4f, and the ICB assay was performed as previously described (Duxin et al., 2009). Images were processed by ImageJ

Software using the grey scale and invert functions, and cell nuclei and ICBs were counted.

Molecular dynamics simulations

The c-MYC Pu24T solution structures (PDB ID: 2MGN) were downloaded from the Protein Data Bank (Chung et al., 2014). Eight c-MYC Pu24T-4f structures were modeled based on various 4f and 8g binding modes using the Openbabel (O'Boyle et al., 2011), Avogadro (Hanwell et al., 2012), and Chimera (Pettersen et al., 2004) software packages. Each complex was placed inside the center of a dodecahedron box, solvated by adding water molecules, and neutralized by adding an excess of 100 mM KC1 using GROMACS tools (Abraham et al., 2015). The DNA was simulated with the

Amber99SB (Homak et al., 2006) force-field parameters with PARMBSC1 (Ivani et al., 2016) improvements, and the tip3p model (Jorgensen et al., 1983) was used for water molecules. Before assigning GAFF force-field parameters (Wang et al., 2004) to 4f and 8g, its partial atomic charges were computed by the RESP method (Bayly et al., 1993) using AmberTool (D.A. Case et al., 2017) after geometry optimization by PM6 and B3LYP/6-31g(d,p) methods in two stages using the Gaussian package (Frisch et al., 2016). Subsequently, MD simulations were performed using GROMACS-2016

(Abraham et al., 2015)) as previously described (Prasad et al., 2018). The obtained MD trajectories were combined and clustered on the basis of principle component analysis using gmx clusterByFeatures (https://gmx-clusterbyfeatures.readthedocs.io). The binding energy was calculated with the MM/PBSA method using the g mmpbsa tool (Baker et al., 2001; Kumari et al., 2014).

Caco-2 cell permeability assay

Caco-2 cell monolayers (passage 94-105) were grown on permeable filter supports and used for the transport study on day 21 after seeding. Prior to the experiment, a drug solution of 10 mM was prepared and warmed to 37°C. The Caco-2 filters were washed with pre-warmed HBSS prior to the experiment, and the experiment was started by applying the donor solution to the apical or basolateral side. The transport experiments were carried out at pH 7.4 in both the apical and basolateral chambers. The experiments were performed at 37°C and with a stirring rate of 500 rpm. The receiver compartment was sampled at 15, 30, and 60 minutes, and at 60 minutes a final sample was also taken from the donor chamber in order to calculate the mass balance of the compound. The samples (100 pi) were transferred to a 96-well plate containing 100 mΐ methanol and warfarin as IS and were sealed until LC-MS/MS analysis.

Cancer cell line screening

Cell lines that have been preserved in liquid nitrogen are thawed and expanded in vendor recommended growth media. Once cells have reached expected doubling times, screening begins. Cells are seeded in growth media in black 384-well tissue culture treated plates at 500-1500 cells per well. Cells are equilibrated in assay plates via centrifugation and placed at 37°C 5% CO2 for twenty -four hours before treatment. At the time of treatment, a set of assay plates (which do not receive treatment) are collected and ATP levels are measured by adding CellTiter-Glo 2.0 (Promega) and luminescence read on Envision plate readers (Perkin Elmer). Assay plates are incubated with compound for 3 days and are then analyzed using CellTiter-Glo 2.0. All data points are collected via automated processes and are subject to quality control and analyzed using Horizon’s proprietary software. Inhibition levels of 0% represent no inhibition of cell growth by treatment. Inhibition of 100% represents no doubling of cell numbers during the treatment window. Both cytostatic and cytotoxic treatments can yield an Inhibition percentage of 100%. Inhibition percentage is calculated as the following:

1=1 -T/U

where T is the treated and U is the untreated/vehicle control. Concentration of KC45 (8g) used were 10, 5, 2.5, 1.25, 0.625, 0.313, 0.16, 0.08, 0.04, and 0 mM.

Viral replicon particles

CHIKV Viral replicon particles (VRPs) were produced as reported by Glasker et al. (2013) except that the structural proteins were replaced by a sequence coding for eGFP. In short, these VRPs are only capable to perform a single-cycle infection and eGFP is expressed in cells upon replication and translation of the viral +ssRNA. Cell culture (BHK cells)

Baby Hamster Kidney (BHK) cells were grown in Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics.

Viral replication assay

BHK cells were seeded in an 8-well IBIDI m-slide one day prior to infection. Cells were treated with MEM containing KC45 (8g) at various concentrations: 0, 2.5, and 5 mM and CHIKV VRPs at a high Multiplicity Of Infection (MOI) to ensure that all cells will get infected. In a control well, cells were only treated with DMSO. The expression of eGFP was assessed 8 hours post-infection using a fluorescence microscope.

KC45 (8g) toxicity assay

BHK cells were seeded in a 96 well plate one day prior to addition of KC45 (8g). Cells were incubated with 0, 0.625, 1.25, 2.5 or 5 mM of KC45 and the survival of the cells was assessed at 17 and 44 hours using AlamarBlue according to the manufacturer protocol. Experiments were conducted with 4 replicates.

EXAMPLES OF THE INVENTION

To improve the hit compounds ability to bind and stabilize G4 DNA structures and to understand which factors that control selectivity and potency, synthetic routes were designed to broadly explore the compounds structure-activity and structure-selectivity relationships. This resulted in a library of forty-seven derivatives that are all based on the initial hit compound 5b, as outlined in Figure 6(a) and scheme 1-5.

The key intermediates 3a-h were synthesized from commercially available substituted anilines (la-h) in two steps. In the first step, a modified Skraup synthesis (Guiles et ak, 2009) was used to generate the substituted 2, 2, 4-trimethyl- 1,2-dihydroquinolines (2a-h) in 53-78 % yield. In the second step, the 1,2-dihydroquinolines (2a-h) were reacted with 2-cyanoguanidine to give A-(4-methyl-quinazolin-2-yl)-guanidine intermediates (3a-h) in 45-57% yields. Finally, treatment of the intermediates 3a-h with acetylacetone yielded the desired quinazoline-pyrimidine derivatives 4(a, c-f) in 50-65% yield (Scheme 1). Intermediate 3(b, g-h) was not compatible with this method and was therefore synthesized using a different approach starting with the synthesis of 4,6- dimethyl-pyrimidin-2-yl-cyanamide (6) from 2-cyanoguanidine and acetylacetone. Condensation of 6 with 2, 2, 4-trimethyl- 1,2-dihydroquinolines 2(b, g-h) under acidic conditions gave the desired quinazoline-pyrimidine derivatives 4(b, g-h) in 23-31% yield (Scheme 2). In addition to these derivatives, the condensation of 3a-b with mesityl oxide in DMSO at 100°C also yielded quinazoline-dihydropyrimidine derivatives (Safa) (Scheme 1) in 21-26% yield.

The compounds 8 (a-g), 9 (a-f) and 12-17 can exist in two keto and one enolic form as shown in Figure 6(b).

Scheme 1. Synthesis of various quinazoline-pyrimidine derivatives; Reagents and conditions: i. dry acetone, h, /-butyl catechol, MgSCri, reflux 16-18 h, (53-78 %); ii. 2-cynogunidine, 2M HC1, 100°C, 0.5 h, (45-57 %); iii. acetylacetone, acetic acid, reflux, 12 h, (51-61 %); iv. mesityl oxide, DMSO, 100 ^°C, 12 h, (21- 26%).

Scheme 2. Synthesis of various quinazoline-pyrimidine derivatives 4(b, g-h); Reagents and conditions: i. 2N NaOH, water, reflux, 16 h, (63%); ii. Dioxane 2 M HC1, 105^°C, 2h, (23-31%).

Condensation of benzalacetone with some of the /V-(4-m ethyl -quinazolin-2-yl)- guanidine derivatives (3a, g) in DMSO has been reported to result in 4-methyl-/V-(4- methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine derivatives (7a, g) (Shikhaliev et ah, 2002). However, this method gave very low yields (19-24%) and, as a consequence, a modified approach was developed using microwave-heating and by varying solvent, temperature, and reaction time. /V-(4-methyl-quinazolin-2-yl)-guanidine derivative 3a did not react with benzalacetone when pyridine or tetrahydrofuran was used as solvent and only a very low conversion (10%) was observed in 1,2-dichloroethane when heated by microwaves at 155°C for 35 min. Instead, polar aprotic solvents worked well;

dimethylformamide and dimethylsolfoxide gave the desired 7a in 10% and 12% yield already after lh at 100°C, which could be further improved to 53% and 61% yield, respectively, after heating at 155°C for 35 min. Substituted 4-methyl-/V-(4-methyl-6- phenylpyrimidin-2-yl)quinazolin-2-amine derivatives 7a-g was thus synthesized in 50- 61% yields starting from 3a-g by using microwave heating at 135°C for 35 min using DMSO as solvent (Scheme 3).

In order to synthesize quinazoline-quinazoline derivatives, a well-known reaction of different isatoic anhydride with /V-(4-m ethyl -quinazolin-2-yl)-guani dine derivatives (3a-h) was performed under basic conditions to give desired compounds 8(a-g) and 9(a- f) in 60-73% yield (Scheme 3).

Scheme 3. Synthesis of various quinazoline-pyrimidine 7(a-g) and quinazoline- quinazoline derivatives 8(a-g) & 9(a-f). Reagents and conditions: i) DMSO, benzalacetone, 100°C, 12 h, (19-21%); ii. DMSO, benzal acetone, Microwave, 135°C, 0.5 h, (50-61%); iii. DMF, different isatoic anhydrides, N,N- diisopropylethylamine, 100°C, 12 h, (60-73%). * means no reaction. Next, 4,6-dimethyl-pyrimidin-2-yl-cyanamide (6) was used as starting material to synthesize pyrimidine-pyrimidine and quinazoline-pyrimidine derivatives by reduction with ammonium chloride under refluxing conditions to give l-(4,6-dimethylpyrimidin- 2-yl)guanidine derivative (10) (Scheme 4). Reacting 10 with acetyl acetone or different substituted isatoic anhydrides (obtained through reaction of substituted anthranilic acid with triphosgene) yielded desired pyrimidine-pyrimidine (11) or quinazoline-pyrimidine derivatives 12-17 in 43-57% yields.

Scheme 4. Synthesis of various pyrimidone derivatives; Reagents and conditions: i) Phenol, NH4CI, 125°C, 4 h, (68%); ii. Acetic acid 125^°C, 12 h

(64%); iii. Different isatoic anhydrides, DMF, A( A-di i sopropyl ethyl ami ne, 100°C, 12 h, (43-57%).

Experimental procedure

All analytical grade reagents and solvents were purchased from Sigma-Aldrich, Fluka, or Acros and used as supplied unless stated otherwise. Thin layer chromatography (TLC) was used for monitoring of chemical reactions and performed on aluminium backed silica gel plates (median pore size 60 A, fluorescent indicator 254 nm) and detected with UV light at 254 and 366 nm. Flash column chromatography was performed using silica gel (0.063-0.200 mesh). Automated flash column

chromatography was performed using a Biotage Isolera One system and purchased prepacked silica gel cartridges (Biotage SNAP Cartridge, KP-Sil). Dimethylformamide (DMF) was dried in a solvent drying system (activated molecular sieves in combination with an isocyanate scrubber). 'H and ¹³C NMR spectra were recorded on Bruker 400 or 600 MHz spectrometers at 298 K and calibrated by using the residual peak of the solvents as the internal standard (DMSO-d₆ : d H = 2.50 ppm; d C = 39.50 ppm and CDCI3 : d H = 7.26 ppm; d C = 77.02 ppm). The coupling constant values (. J) are determined in Hertz. The abbreviations used in NMR data are mentioned as, singlet = s, doublet = d, triplet = t, multiplet = m, double doublet = dd, and broad singlet = brs. LC- MS was conducted on an Agilent 6150 Series Quadrupole LC/MS system. HRMS was performed by using an Agilent 1290 binary LC system connected to an Agilent 6230 Accurate-Mass TOF LC/MS (ESI+); calibrated with an Agilent G1969-85001 ES-TOF Reference Mix containing ammonium trifluoroacetate, purine and h ex ak i s( 1 //, 1 //, 3 H- tetrafluoropropoxy)phosphazine in 90 : 10 acetonitrile : water. Preparatory HPLC was performed with a Gilson instrument using a Nucleodur C18 HTec reversed-phase column (25 cm x 21.5 mm; particle size 5 pm) with H O/MeCN mixtures as the eluent. Microwave reactions were carried out in an Initiator+ microwave instrument from Biotage, using sealed 0.2-0.5 mL and 10-20 mL process vials. Reaction times refer to irradiation time at the target temperature, not the total irradiation time. The temperature was measured with an IR sensor.

General procedure for the synthesis of 2,2,4-trimethyl-l,2-dihydroquinoline derivatives 2(a-h): The mixture of different anilines l(a-h) (12.17 mmol) with anhydrous magnesium sulphate (7.3 g, 60.67 mmol) in anhydrous acetone (50 ml) was added to iodine (154mg, 5 mol%) and /er/-butyl catechol (61 mg, 3 mol%) and heated to reflux for 12 h. The progress of reaction was monitored on TLC till consumption of aniline and then reaction mixture was allowed to cool and filtered through a bed of celite. The filtrate solution so obtained was concentrated under reduced pressure to give brown coloured semi-solid material which was purified through column

chromatography over silica in EtOAc (0.5-5%) in heptane to give desired 2,4-trimethyl- 1,2-dihydroquinoline derivatives 2(a-h).

6-Methoxy-2,2,4-trimethyl-l,2-dihydroquinoline (2a): The title compound (2a) was obtained from the reaction of /i-anisidine with acetone as a brown oil in 71% yield by following the general procedure. ¹H NMR (400 MHz, CDCL), d (ppm): 6.72 (d, J= 4.0 Hz, 1H), 6.63 (dd, J= 4.0 & 8.0 Hz, 1H), 6.42 (d, J= 4.0 Hz, 1H), 5.39 (s, 1H), 3.76 (s, 3H), 2.00 (s, 3H), 1.26 (s, 6H); ¹³C NMR (100 MHz, CDCL), d (ppm): 152.03, 137.53, 129.79, 128.54, 122.98, 113.70, 113.51, 110.10, 55.90, 51.73, 30.36, 18.62; ESI MS (m/z): calculated for C H_ISNO (M+H)⁺: 204.1383, found 204.3. 7-Methoxy-2,2,4-trimethyl-l,2-dihydroquinoline (2b): The title compound (2b) was obtained from the reaction of w-anisidine with acetone as a brown oil in 78% yield by following the general procedure. ¾ NMR (400 MHz, CDCh), d (ppm): 6.97 (d, J= 8.0 Hz, 1H), 6.20 (dd, J= 8.0 & 4.0 Hz, 1H), 6.01 (d, J= 4.0 Hz, 1H), 5.19 (s, 1H), 3.75 (s, 3H), 1.96 (s, 3H), 1.26 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 160.17, 144.60, 128.21, 125.93, 124.62, 115.36, 115.36, 102.27, 98.59, 55.10, 51.93, 31.08, 18.62; ESI MS (m/z): calculated for CnHisNO (M+H)⁺: 204.1383, found 204.2.

6.7-Dimethoxy-2,2,4-trimethyl-l,2-dihydroquinoline (2c): The title compound (2c) was obtained from the reaction of 4-aminoveratrole with acetone as a brown oil in 67% yield by following the general procedure. ¾ NMR (400 MHz, CDCh), d (ppm): 6.67 (s, 1H), 6.08 (s, 1H), 5.20 (s, 1H), 3.80, 3.81 (2s, 6H), 1.96 (s, 3H), 1.24 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 149.67, 141.04, 138.12, 128.28, 126.28, 114.02, 109.25, 98.16, 57.14, 55.80, 51.81, 30.50, 18.71; ESI MS (m/z): calculated for

C14H20NO2 (M+H)⁺: 234.1489, found 234.2.

5.6.7-Trimethoxy-2,2,4-trimethyl-l,2-dihydroquinoline (2d): The title compound (2d) was obtained from the reaction of 3,4,5-trimethoxyaniline with acetone as a brown oil in 76% yield by following the general procedure. ¾ NMR (400 MHz, CDCh), d (ppm): 5.88 (s, 1H), 5.17 (s, 1H), 3.83, 3.79, 377 (3s, 9H), 2.15 (s, 3H), 1.22 (s, 6H);

¹³C NMR (100 MHz, CDCh), d (ppm): 153.19, 152.01, 141.01, 134.60, 128.65, 127.98, 114.83, 108.61, 93.74, 61.11, 60.97, 55.71, 29.69, 22.14; ESI MS (m/z): calculated for C15H22NO3 (M+H)⁺: 264.1594, found 264.2.

2,2,4-Ttrimethyl-l,2-dihydrobenzo[/ ]quinolin-7-ol (2e): The title compound (2e) was obtained from the reaction of 5-aminonaphthalen-l-ol (le) with acetone as a brown oil in 53% yield by following the general procedure. ¾ NMR (400 MHz, CDCh), d (ppm): 7.43 (d, J= 8.0 Hz, 1H), 7.29-7.34 (m, 2H), 7.23 (t, J= 8.0 Hz, 1H), 6.76 (d, J= 8.0 Hz, 1H), 5.36 (s, 1H), 5.21 (brs, 1H), 4.47 (brs, 1H), 2.10 (s, 3H), 1.36 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 153.67, 139.38, 128.51, 126.89, 125.47, 125.60, 124.19, 123.32, 121.25, 114.52, 113.00, 109.06, 108.29, 31.17, 19.29; ESI MS (m/z): calculated for CieHisNO (M+H)⁺: 240.1383, found 240.3. 2.2.4-Trimethyl-l,2,7,8,9,10-hexahydrobenzo[/ ]quinoline (2f): The title compound (2f) was obtained from the reaction of 5,6,7,8-tetrahydro-l-naphthylamine (If) with acetone as a brown solid in 59% yield by following the general procedure. 'H NMR (400 MHz, CDCh), d (ppm): 6.90 (d, J= 8.0 Hz, 1H), 6.42 (d, J= 8.0 Hz, 1H), 5.25 (s, 1H), 2.70 (t, 2H, J= 8.0 Hz), 2.37 (t, J= 8.0 Hz, 2H), 1.98 (s, 3H), 1.83-1.89 (m, 2H), 1.70-1.76 (m, 2H), 1.28 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 140.67, 137.40, 128.88, 126.52, 120.80, 118.83, 118.26, 111.27, 51.73, 31.52, 30.14, 23.58, 23.13, 22.71, 18.82; ESI MS (m/z): calculated for Ci₆H₂₂N (M+H)⁺: 228.1747, found 228.3.

2.2.4-Trimethyl-l,2-dihydroquinoline (2g): The title compound (2g) was obtained from the reaction of aniline (lg) with acetone as a brown oil in 68% yield by following the general procedure. ¹H MR (400 MHz, CDCh), d (ppm): 7.07 (d, J= 4.0 Hz, 1H), 6.99 (t, J= 8.0 Hz, 1H), 6.65 (t, J= 8.0 Hz, 1H), 6.45 (d, J= 4.0 Hz, 1H), 5.32 (s, 1H), 2.00 (s, 3H), 1.29 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 143.15, 128.56,

128.37, 128.35, 123.62, 121.59, 117.23, 113.00, 51.84, 31.00, 18.61; ESI MS (m/z): calculated for C_I2H_I6N (M+H)⁺: 174.1277, found 174.3.

4-(2,2,4-trimethyl-l,2-dihydroquinolin-6-yl)morpholine (2h): The title compound (2h) was obtained from the reaction of 4-morpholinoaniline (lh) with acetone as a brown oil in 63% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 6.59-6.62 (m, 2H), 6.40 (d, J= 8.0 Hz, 1H), 5.39 (d, J= 4.0 Hz, 1H), 5.29 (s, 1H), 3.70 (t, J= 4.0 Hz, 4H), 2.89 (t, J= 4.0 Hz, 2H), 1.89 (s, 3H), 1.16 (s, 6H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 142.57, 139.07, 129.58, 128.25,

121.37, 117.83, 113.34, 112.94, 66.85, 51.35, 30.84, 18.78; ESI MS (m/z): calculated for C_I6H₂₃N₂0 (M+H)⁺: 259.1805, found 259.2.

General procedure for the synthesis of l-(4-methylquinazolin-2-yl)guanidine derivatives (3a-h): To the mixture of 2, 2, 4-trimethyl- 1,2-dihydroquinoline (0.5g, 2.88 mmol) in 1.1 ml of 2M HC1 solution, 2-cynoguanidine (0.291 g, 3.64 mmol) was added and heated at 105°C for 15-40 mins. Upon cooling a precipitation was formed which was collected through filtration. Finally, the precipitate so obtained were sonicated in 5 ml of methanol (20%) and ammonia (20%) in water for 15 min and filtered, washed (lml of methanol), and dried under vacuum to give pure l-(4-methylquinazolin-2- yl)guanidine derivatives (3a-h) in 47-55% yield. l-(6-Methoxy-4-methylquinazolin-2-yl)guanidine (3a): The title compound (3a) was obtained from the reaction of 2a with 2-cynoguanidine as a white solid in 56% yield by following the general procedure. 'H NMR (400 MHz, DMSO-<¾), d (ppm): 11.08 (s, 1H), 8.85 (brs, 3H), 7.86 (d, J= 8.0 Hz, 1H), 7.59 (dd, J= 4.0 & 8.0 Hz, 1H), l-(6- Methoxy-4-methylquinazolin-2-yl)guanidine (3a) 7.44 (d, ./ = 4.0 Hz, 1H), 3.92 (s, 3H), 2.86 (s, 3H); ¹³C NMR (100 MHz, DMSO-i¾), d (ppm): 170.81, 157.51, 155.69, 152.18, 144.50, 128.70, 127.68, 122.33, 104.73, 56.33, 22.15; ESI MS (m/z): calculated for C₁₁H₁₄N₅O (M+H)⁺: 232.1193, found 232.2. l-(7-Methoxy-4-methylquinazolin-2-yl)guanidine (3b): The title compound (3b) was obtained from the reaction of 2b with 2-cynoguanidine as a white solid in 53% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 7.98 (d, ./ = 8.0 Hz, 1H), 7.16 (d, = 4.0 Hz, 1H), 7.03 (dd, J= 4.0 & 8.0 Hz, 1H), 3.91 (s, 3H), 2.73 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 169.02, 164.13, 159.83, 158.40, 152.41, 127.64, 116.76, 115.48, 105.49, 56.12, 21.77; ESI MS (m/z): calculated for C₁₁H₁₄N₅O (M+H)⁺: 232.1193, found 232.2. l-(6,7-Dimethoxy-4-methylquinazolin-2-yl)guanidine (3c): The title compound (3c) was obtained from the reaction of 2c with 2-cynoguanidine as a white solid in 57% yield by following the general procedure. 'H NMR (400 MHz, DMSO-ίL;), d (ppm): 8.01 (brs, 1H), 7.30 (s, 1H), 7.20 (s, 1H), 3.91, 3.93 (2s, 6H), 2.74 (s, 3H); ¹³C NMR (100 MHz, DMSO-£¾), d (ppm): 167.15, 157.93, 156.16, 148.14, 147.42, 115.21, 105.92,

104.17, 56.40, 56.29, 21.88; ESI MS (m/z): calculated for C12H16N5O2 (M+H)⁺:

262.1299, found 262.2. l-(5,6,7-trimethoxy-4-methylquinazolin-2-yl)guanidine (3d): The title compound (3d) was obtained from the reaction of 2d with 2-cynoguanidine as a white solid in 55% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i/g), d (ppm):

7.61 (brs, 3H), 6.95 (s, 1H), 3.92, (s, 6H), 3.79 (s, 3H), 2.78 (s, 3H); ¹³C NMR (100 MHz, DMSO-i/g), d (ppm): 166.95, 161.80, 159.76, 159.25, 150.53, 149.85, 139.72, 110.48, 102.85, 61.93, 61.53, 56.88, 26.88; ESI MS (m/z): calculated for C₁₃H₁₈N₅O₃ (M+H)⁺: 292.1404, found 292.2. l-(7-Hydroxy-4-methylbenzo[h]quinazolin-2-yl)guanidine (3e): The title compound (3e) was obtained from the reaction of 2e with 2-cynoguanidine as a white solid in 45% yield by following the general procedure. 'H NMR (400 MHz, DMSO-<¾), d (ppm):

8.26 (d, J= 8.0 Hz, 1H), 7.89 (d, J= 8.0 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.14 (d, J= 8.0 Hz, 1H), 2.77 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 167.53, 163.45, 159.80, 154.01, 150.09, 130.76, 127.50, 125.87, 120.78, 117.57, 115.97, 114.99, 113.03, 22.31; ESI MS (m/z): calculated for C14H14N5O (M+H)⁺:

268.1193, found 268.2. l-(4-Methyl-7,8,9,10-tetrahydrobenzo[/ ]quinazolin-2-yl)guanidine (3f): The title compound (3f) was obtained from the reaction of 2f with 2-cynoguanidine as a white solid in 54% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-<¾), d (ppm): 8.27 (brs, 3H), 7.82 (d, J= 8.0 Hz, 1H), 7.16 (d, J= 8.0 Hz, 1H), 2.92 (t, J = 8.0 Hz, 2H), 2.86 (t, J= 8.0 Hz, 2H), 2.76 (s, 3H), 1.84-1.86 (m, 2H), 1.78-1.80 (m, 2H);¹³C NMR (100 MHz, DMSO-^), d (ppm): 170.21, 158.16, 148.48, 143.47, 131.44, 126.68, 122.76, 118.43, 30.25, 24.27, 22.64, 22.54, 21.90; ESI MS (m/z): calculated for C_MH_ISN_S (M+H)⁺: 256.1557, found 256.2. l-(4-Methylquinazolin-2-yl)guanidine (3g): The title compound (3g) was obtained from the reaction of 2g with 2-cynoguanidine as a white solid in 51% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 7.97 (d, ./ = 8.0 Hz, 1H), 7.69 (t, J= 8.0 Hz, 1H), 7.57 (d, J= 8.0 Hz, 1H), 7.49 (brs, 2H), 7.29 (t, J = 8.0 Hz, 1H), 2.73 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 169.58, 161.52, 159.30, 150.13, 134.00, 126.29, 125.98, 123.97, 119.89, 22.00; ESI MS (m/z):

calculated for C10H12N5 (M+H)⁺: 202.1087, found 202.2. l-(4-Methyl-6-morpholinoquinazolin-2-yl)guanidine (3h): The title compound (3h) was obtained from the reaction of 2h with 2-cynoguanidine as a white solid in 50% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 7.67 (d, J= 8.0 Hz, 1H), 7.60 (d, J= 8.0 Hz, 1H), 7.19 (s, 1H), 3.78 (t, J= 4.0 Hz, 4H), 3.22 (t, J= 4.0 Hz, 2H), 2.74 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm):

168.24, 158.67, 158.30, 147.94, 144.81, 127.23, 126.32, 120.71, 107.07, 66.53, 49.26, 22.06; ESI MS (m/z): calculated for C_I4H_I9N₆0 (M+H)⁺: 287.1615, found 287.3. General procedure for the synthesis of /V-(4,6-dimethylpyrimidin-2-yl)-4- methylquinazolin-2-amine derivatives (4a, c-f): The mixture of l-(4- methylquinazolin-2-yl)guanidine derivatives (4a, c-f) (0.39 mmol) and acetylacetone (0.78 mmol) in acetic acid (0.5 mL) was heated to reflux for 12 h. Progress of the reaction was monitored on TLC. On completion, the reaction mixture was basified (pH 8-9) with 28% ammonium hydroxide solution to give a yellow precipitate which was filtered, dried, and purified through column chromatography over silica gel using MeOH (0-2%) and triethylamine (0.2%) in DCM to give desired L-(4,6- dimethylpyrimidin-2-yl)-4-methylquinazolin-2-amine derivatives (4a, c-f) in 50-61 % yield.

/V-(4,6-Dimethylpyrimidin-2-yl)-6-methoxy-4-methylquinazolin-2-amine (4a)

(ECH-69): The title compound (4a) was obtained from the reaction of 3a with acetylacetone as a light yellow solid in 61% yield by following the general procedure. ¾ NMR (400 MHz, CDCh), d (ppm): 8.03 (s, 1H), 7.82 (d, J= 8.0 Hz, 1H), 7.43 (dd, J = 4.0 & 8.0 Hz, 1H), 7.18 (d, J= 4.0 Hz, 1H), 6.66 (s, 1H), 3.92 (s, 3H), 2.84 (s, 3H), 2.46 (s, 6H); ¹³C NMR (150 MHz, CDCh), d (ppm): 168.14, 168.01, 158.50, 156.32, 153.45, 146.88, 129.40, 125.75, 121.51, 113.81, 103.05, 55.58, 24.12, 21.86; HRMS: (m/z) calcd for CieHisNsO [M+H]⁺: 296.1506, found 296.1506.

/V-(4,6-Dimethylpyrimidin-2-yl)-6,7-dimethoxy-4-methylquinazolin-2-amine (4c)

(ECH-71): The title compound (4c) was obtained from the reaction of 3c with acetylacetone as a light-yellow solid in 57% yield by following the general procedure.

¾ NMR (400 MHz, CDCh), d (ppm): 8.14 (s, 1H), 7.24 (s, 1H), 7.11 (s, 1H), 6.64 (s, 1H), 3.99 (s, 3H), 4.01 (s, 3H), 2.78 (s, 3H), 2.45 (s, 6H); ¹³C NMR (100 MHz, CDCh), d (ppm): 168.02, 166.60, 158.50, 155.61, 154.07, 148.81, 148.05, 115.89, 113.76, 106.96, 102.74, 56.32, 56.04, 24.15, 21.74; HRMS: (m/z) calcd for C17H20N5O2

[M+H]⁺: 326.1612, found 326.1615.

/V-(4,6-dimethylpyrimidin-2-yl)-5,6,7-trimethoxy-4-methylquinazolin-2-amine 2- ((4,6-Dimethylpyrimidin-2-yl)amino)-4-methylbenzo \h\ quinazolin-7-ol (4d) (ECH- 72): The title compound (4d) was obtained from the reaction of 3d with acetylacetone as a light yellow solid in 50% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 10.31 (s, 1H), 10.04 (s, 1H), 8.56 (dd, J= 4.0 & 8.0 Hz, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.89 (d, J= 8.0 Hz, 1H),7.50 (t, J= 8.0 Hz, 1H), 7.16 (dd, J= 8.0 Hz, 1H), 6.85 (s, 1H), 2.85 (s, 3H), 2.39 (s, 6H); ¹³C NMR (100 MHz, DMSO- d/), d (ppm): 168.34, 167.51, 159.24, 155.88, 153.79, 150.70, 131.44, 127.70, 125.77, 120.58, 119.18, 117.80, 115.93, 114.06, 113.47, 24.02, 21.95; HRMS: (m/z) calcd for CigHisNsO [M+H]⁺: 332.1506, found 332.1506.

2-((4,6-Dimethylpyrimidin-2-yl)amino)-4-methylbenzo \h\ quinazolin-7-ol (4e or KC13) (ECH-73): The title compound (4e) was obtained from the reaction of 3e with acetylacetone as a light yellow solid in 50% yield by following the general procedure. ¾ NMR (400 MHz, CDCI3), d (ppm): 7.97 (s, 1H), 7.06 (s, 1H), 6.66 (s, 1H), 4.00 (s, 3H), 3.99 (s, 3H), 3.91 (s, 3H), 2.94 (s, 3H), 2.46 (s, 6H); ¹³C NMR (100 MHz, CDCI3), d (ppm): 168.04, 167.95, 158.94, 158.38, 154.35, 150.26, 150.18, 140.19, 113.92, 111.85, 103.33, 61.13, 61.10, 56.19, 26.26, 24.14; HRMS: (m/z) calcd for C18H22N5O3 [M+H]⁺: 356.1717, found 356.1720.

/V-(4,6-Dimethylpyrimidin-2-yl)-4-methyl-7,8,9,10-tetrahydrobenzo[h]quinazolin-

2-amine (4f or KC29) (ECH-74): The title compound (4f) was obtained from the reaction of 3f with acetylacetone as a light yellow solid in 50% yield by following the general procedure. ¾ NMR (400 MHz, CDCI3), d (ppm): 8.11 (s, 1H), 7.69 (d, J= 8.0 Hz, 1H), 7.09 (d, J= 8.0 Hz, 1H), 6.67 (s, 1H), 3.25 (t, J= 8.0 Hz, 2H), 2.89 (t, J= 8.0 Hz, 2H), 2.83 (s, 3H), 2.47 (s, 6H), 1.86-1.93 (m, 4H); ¹³C NMR (100 MHz, CDCI3), d (ppm): 169.34, 167.80, 158.54, 154.04, 150.24, 142.75, 133.72, 126.47, 121.56, 119.10, 113.65, 30.43, 24.26, 24.11, 22.83, 22.80, 21.78; HRMS: (m/z) calcd for C19H22N5

[M+H]⁺: 320.1870, found 320.1872.

Synthesis procedure for synthesis of /V-(4,6-dimethylpyrimidin-2-yl)cyanamide (6): To the mixture of 2-cyanoguanidine (5g, 59.46 mmol) and acetylacetone (9.2 mL, 89.20 mmol) in water (37 mL), 2.85 mL of 2M of NaOH was added and the reaction mixture was heated at 105°C for 24 h. Progress of the reaction mixture was monitored through TLC. On completion, the reaction mixture was cooled to room temperature which resulted in precipitation of the product. Filtration and washing of the precipitate with ethanol gave a light-yellow solid in 63% yield. ¾ NMR (400 MHz, DMSO- is), d (ppm): 12.57 (brs, 1H), 6.64 (s, 1H), 2.31 (s, 6H); ¹³C NMR (100 MHz, DMSO-^e), d (ppm): 167.20, 160.69, 116.25, 110.15, 22.26; ESI MS (m/z): calculated for C7H9N4 (M+H)⁺: 149.0773, found 149.1.

General procedure for the synthesis of /V-(4,6-dimethylpyrimidin-2-yl)-4- methylquinazolin-2-amine derivatives 4(b,g-h): To the mixture of 2,2,4-trimethyl- 1,2-dihydroquinoline 2(b, g-h, (150 mg, 0.86 mmol)) and /V-(4,6-dimethylpyrimidin-2- yl)cyanamide (6 (128.3 mg, 0.86 mmol)) in dioxane (5 mL), 2M HC1 (455 pL) was added and the reaction mixture was heated at 105°C for two h. Progress of the reaction mixture was monitored with TLC. Upon consumption of all starting material, reaction mixture was cooled to room temperature, basified with ammonia and concentrated under reduced pressure. Finally, the crude reaction mixture is purified through HPLC in Gilson instrument with acetonitrile (10-70%) and TFA (0.1%) in water system to give required derivatives in 23-31% yield.

/V-(4,6-dimethylpyrimidin-2-yl)-7-methoxy-4-methylquinazolin-2-amine (4b)

(ECH-70): The title compound (4b) was obtained from the reaction of 2b with 6 as a light yellow solid in 26% yield by following the general procedure. ¾ NMR (400 MHz, CDCE), d (ppm): 8.79 (s, 1H), 7.74 (d, J= 8.0 Hz, 1H), 7.17 (d, J= 4.0 Hz, 1H), 6.91 (dd, J= 4.0 & 8.0 Hz, 1H), 6.58 (s, 1H), 3.84 (s, 3H), 2.71 (s, 3H), 2.39 (s, 6H);

¹³C NMR (100 MHz, CDCE), d (ppm): 168.56, 167.95, 163.80, 158.51, 155.33, 153.64, 126.23, 117.17, 116.21, 113.88, 106.17, 55.56, 24.05, 21.44; HRMS: (m/z) calcd for CieHisNsO [M+H]⁺: 296.1506, found 296.1506.

/V-(4,6-Dimethylpyrimidin-2-yl)-4-methylquinazolin-2-amine (4g) (ECH-75): The title compound (4g) was obtained from the reaction of 2g with 6 as a light yellow solid in 31% yield by following the general procedure. ¾ NMR (400 MHz, CDCE), d (ppm): 8.10 (brs, 1H), 7.96 (d, J= 8.0 Hz, 1H), 7.89 (d, J= 8.0 Hz, 1H), 7.75 (t, J= 8.0 Hz, 1H), 7.40 (t, J= 8.0 Hz, 1H), 6.68 (s, 1H), 2.87 (s, 3H), 2.48 (s, 6H); ¹³C NMR (100 MHz, CDCE), d (ppm): 170.01, 168.08, 158.32, 154.56, 151.09, 133.66, 127.94,

124.98, 124.63, 121.10, 114.13, 24.14, 21.74; HRMS: (m/z) calcd for C_I5H_I6N₅

[M+H]⁺: 266.1400, found 266.1404.

/V-(4,6-dimethylpyrimidin-2-yl)-4-methyl-6-morpholinoquinazolin-2-amine (4h)

(ECH-76): The title compound (4h) was obtained from the reaction of 2h with 6 as a yellow solid in 23% yield by following the general procedure. 'H NMR (400 MHz, CDC ), d (ppm): 8.03 (s, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.54 (dd, J= 4.0 & 8.0 Hz, 1H), 7.13 (d, J= 4.0 Hz, 1H), 6.65 (s, 1H), 3.91 (t, J= 4.0 Hz, 4H), 3.25 (t, J= 4.0 Hz, 4H), 2.82 (s, 3H), 2.46 (s, 6H); ¹³C NMR (150 MHz, CDCh), d (ppm): 168.13, 168.00, 158.48, 153.25, 148.06, 146.66, 128.80, 126.07, 121.68, 113.76, 106.78, 66.84, 49.68, 24.14, 21.84; HRMS: (m/z) calcd for CifcNeO [M+H]⁺: 351.1928, found 351.1930.

General procedure for synthesis of 4-methyl-/V-(4,4,6-trimethyl-4,5- dihydropyrimidin-2-yl)quinazolin-2-amine derivatives (5a-b): A mixture of quinazolylguanidine 3a-b (500 mg 2.16 mmol) and mesityl oxide (330 pL, 2.87 mmol) in DMSO (2 ml) was heated at 100°C for 12 h. Upon consumption of the starting material, the reaction mixture was cooled down to room temperature and poured into ice cold water and extracted with DCM. The organic layer were then dried over anhydrous Na₂SC>₄ and concentrated under reduced pressure to give the crude mixture, which was recrystallized with acetone to give the desired products in 21-26% yield.

6-Methoxy-4-methyl-/V-(4,4,6-trimethyl-l,4-dihydropyrimidin-2-yl)quinazolin-2- amine (5a) (ECH-77): The title compound (5a) was obtained from the reaction of 3a with mesityl oxide as a light yellow solid in 21% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 9.88 (s, 1H), 7.54-7.55 (m, 1H), 7.40 (d, J= 4.0 & 8.0, 1H), 7.31 (d, 1H), 3.88 (s, 3H), 2.73 (s, 3H), 1.72 (s, 2H), 1.32 (s, 6H); ESI MS (m/z): calculated for C11H14N5O (M+H)⁺: 311.1746, found 312.19.

7-Methoxy-4-methyl-/V-(4,4,6-trimethyl-l,4-dihydropyrimidin-2-yl)quinazolin-2- amine (5b) (ECH-78): The title compound (5b) was obtained from the reaction of 3b with mesityl oxide as a light yellow solid in 26% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 9.90 (s, 1H), 7.87 (d, J= 8.0 Hz, 1H), 6.90-6.93 (m, 2H), 3.90 (s, 3H), 2.65 (s, 3H), 1.78 (s, 2H), 1.33 (s, 6H); ESI MS (m/z): calculated for C11H14N5O (M+H)⁺: 311.1746, found 312.21.

General procedure for the synthesis of 4-methyl-N-(4-methyl-6-phenylpyrimidin-2- yl)quinazolin-2-amine 7(a-g): The mixture of l-(4-methylquinazolin-2-yl)guanidine (lOOmg, 0.50 mmol) and benzalacetone (80 mg, 0.55 mmol) in DMSO (0.5 ml) was heated in a sealed tube at 155°C for 35 min using microwaves. The brown colored reaction mixture was then poured into cold brine and extracted with DCM. The organic layer was dried over anhydrous NaiSCri and concentrated under reduced pressure. The crude mixture was then purified through column chromatography over silica using MeOH (0-2%) and triethylamine (0.2%) in DCM to give the desired compounds 7(a-g) in 53-61 % yields.

6-Methoxy-4-methyl-/V-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine (7a) (ECH-79): The title compound (7a) was obtained from the reaction of 3a with benzalacetone as a yellow solid in 61% yield by following the general procedure. 'H NMR (400 MHz, CDCf), d (ppm): 8.18-8.20 (m, 2H), 8.12 (brs, 1H), 7.86 (d, J= 8.0 Hz, 1H), 7.45-7.52 (m, 4H), 7.26 (s, 1H), 7.21 (d, J= 4.0 Hz, 1H), 3.94 (s, 3H), 2.88 (s, 3H), 2.59 (s, 3H, C¾); ¹³C NMR (100 MHz, CDCI3), d (ppm): 169.07, 168.06, 164.78, 158.82, 156.40, 153.49, 146.93, 137.12, 130.67, 129.39, 128.77, 127.24, 125.92,

121.58, 109.69, 103.10, 55.63, 24.62, 21.92; HRMS: (m/z) calcd for C21H20N5O

[M+H]⁺: 358.1662, found 358.1664.

7-methoxy-4-methyl-N-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine (7b or KC18) (ECH-80): The title compound (7b) was obtained from the reaction of 3b with benzalacetone as a yellow solid in 57% yield by following the general procedure. ¾ NMR (400 MHz, CDCI3), d (ppm): 8.16-8.19 (m, 3H), 7.87 (d, J= 8.0 Hz, 1H), 7.48-7.52 (m, 3H), 7.23-7.24 (m, 2H), 7.03 (dd, J= 4.0 & 8.0 Hz, 1H), 3.97 (s, 3H), 2.85 (s, 3H), 2.61 (s, 3H); ¹³ C NMR (100 MHz, CDCI3), d (ppm): 169.18, 168.65, 164.79, 164.04, 158.72, 155.22, 153.66, 137.04, 130.71, 128.76, 127.23, 126.44,

117.40, 116.37, 109.93, 106.10, 55.65, 24.68, 21.63; HRMS: (m/z) calcd for C21H20N5O [M+H]⁺: 358.1662, found 358.1659.

6,7-dimethoxy-4-methyl-N-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine (7c or KC30) (ECH-81): The title compound (7c) was obtained from the reaction of 3c with benzalacetone as a yellow solid in 60% yield by following the general procedure. ¾ NMR (400 MHz, CDCI3), d (ppm): 8.15-8.18 (m, 3H), 7.48-7.50 (m, 3H), 7.27 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 4.05, 4.02 (2s, 6H), 2.85 (s, 3H), 2.61 (s, 3H, C¾); ¹³C NMR (100 MHz, CDCI3), d (ppm): 169.14, 166.52, 164.75, 158.88, 155.72, 154.16, 148.92, 148.15, 137.10, 130.65, 128.74, 127.21, 116.00, 109.67, 106.81, 102.81, 56.29, 56.09, 24.69, 21.81; HRMS: (m/z) calcd for C₂₂H₂₂N₅O₂ [M+H]⁺: 388.1768, found 388.1776.

5,6,7-trimethoxy-4-methyl-N-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2- amine (7d or KC25) (ECH-82): The title compound (7d) was obtained from the reaction of 3d with benzalacetone as a yellow solid in 55% yield by following the general procedure. ¾ NMR (400 MHz, CDCI3), d (ppm): 8.15-8.18 (m, 3H), 7.48-7.51 (m, 3H), 7.22 (s, 1H), 7.08 (s, 1H), 4.01, 4.02, 3.93 (3s, 9H), 2.99 (s, 3H), 2.59 (s, 3H); ¹³C NMR (100 MHZ, CDCh), d (ppm): 169.13, 167.88, 164.73, 159.00, 158.78, 154.48, 150.33, 150.23, 140.23, 137.07, 130.67, 128.73, 127.23, 111.95, 109.77, 103.16, 61.15, 61.13, 56.15, 26.31, 24.67; HRMS: (m/z) calcd for C23H24N5O3 [M+H]⁺: 418.1874, found 418.1876.

4-Methyl-2-((4-methyl-6-phenylpyrimidin-2-yl)amino)benzo[/ ]quinazolin-7-ol (7e) (ECH-83): The title compound (7e) was obtained from the reaction of 3e with benzalacetone as a yellow solid in 51% yield by following the general procedure. 'H NMR (400 MHz, CDCI3), d (ppm): 8.93 (d, J= 8.0 Hz, 1H), 8.31 (brs, 1H), 8.24-8.27 (m, 2H), 8.05 (d, J= 8.0 Hz, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.49-7.54 (m, 4H), 7.28 (s, 1H), 7.10 (d, J= 8.0 Hz, 1H), 2.95 (s, 3H), 2.63 (s, 3H); ¹³C NMR (100 MHz, DMSO- d₆), d (ppm): 169.08, 168.46, 163.72, 159.54, 155.88, 153.89, 150.72, 137.21, 131.38, 131.30, 129.17, 128.83, 120.65, 119.35, 117.93, 115.90, 113.56, 109.98, 24.37, 22.01; HRMS: (m/z) calcd for C24H20N5O [M+H]⁺: 394.1662, found 394.1657.

4-Methyl-/V-(4-methyl-6-phenylpyrimidin-2-yl)-7,8,9,10- tetrahydrobenzo[/i]quinazolin-2-amine (7f or KC31) (ECH-84): The title compound (7f) was obtained from the reaction of 3f with benzalacetone as a yellow solid in 57% yield by following the general procedure. ¹H MR (400 MHz, CDCI3), d (ppm): 8.23- 8.25 (m, 2H), 8.19 (brs, 1H), 7.72 (d, J= 8.0 Hz, 1H), 7.49-7.51 (m, 3H), 7.23 (s, 1H), 7.12 (d, J= 8.0 Hz, 1H), 3.38 (t, J= 8.0 Hz, 2H), 2.92 (t, J= 8.0 Hz, 2H), 2.87 (s, 3H), 2.58 (s, 3H), 1.89-1.97 (m, 4H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 169.28, 168.81, 164.85, 158.85, 154.04, 150.31, 142.75, 137.28, 133.72, 130.61, 128.64,

127.39, 126.53, 121.63, 119.14, 109.68, 99.98, 30.48, 24.75, 24.54, 22.87, 22.82, 21.78; HRMS: (m/z) calcd for C₂₄H₂₄N₅ [M+H]⁺: 382.2026, found 382.2031. 4-Methyl-/V-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2-amine (7g) (ECH-85): The title compound (7g) was obtained from the reaction of 3g with benzalacetone as a yellow solid in 55% yield by following the general procedure. 'H NMR (400 MHz, CDCh), d (ppm): 8.23 (brs, 1H), 8.19-8.21 (m, 2H), 7.98 (d, J= 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.79 (t, J= 8.0 Hz, 1H), 7.49-7.53 (m, 3H), 7.43 (t, J= 8.0 Hz, 1H), 7.25 (s, 1H), 2.92 (s, 3H), 2.60 (s, 3H); ¹³C NMR (100 MHz, CDCh), d (ppm): 169.92, 169.13, 164.82, 158.69, 154.64, 151.15, 137.05, 133.75, 128.79, 127.92, 127.25,

125.06, 124.68, 121.18, 109.96, 24.61, 21.77; HRMS: (m/z) calcd for C20H18N5

[M+H]⁺: 328.1557, found 328.1560.

General procedure for the synthesis of 2-((4-methylquinazolin-2- yl)amino)quinazolin-4(Li/)-one 8(a-g) & 9(a-f): To the mixture of l-(4- methylquinazolin-2-yl)guanidine derivatives 3(a-f) (100 mg 0.50 mmol) and different isatoic anhydride (97 mg, 0.60 mmol) in anhydrous DMF (1 mL), diisopropyl ethylamine (103 pL, 0.60 mmol) was added and the reaction mixture was heated at 100°C for 12 h. The reaction became a clear solution after stirring for 15 min and reprecipitation of the product appeared after 8 h, however, the reaction was continued until consumption of all starting material. On completion, the reaction mixture was allowed to cool to room temperature. The precipitate was filtered, washed with methanol, and purified through crystallization in methanol to give the desired 2-((4- m ethyl quinazolin-2-yl)amino)quinazolin-4( l H)-one 8(a-g) and 9(a-f) in 60-73% yield.

2-((6-Methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(LiF)-one (8a or KC41)

(ECH-86): The title compound (8a) was obtained from the reaction of 3a with isatoic anhydride, as a white solid in 67% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 13.56 (s, 1H), 10.94 (s, 1H), 8.06 (d, J= 8.0 Hz, 1H), 7.76 (d, J= 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.63 (dd, J = 4.0 & 8.0 Hz, 1H), 7.50 (d, J= 4.0 Hz, 1H), 7.48 (d, 7= 8.0 Hz, 1H), 7.31 (t, 7= 8.0 Hz, 1H), 3.95 (s, 3H), 2.91 (s, 3H); ¹³C NMR (100 MHz, DMSO-7;), d (ppm): 170.40, 157.00, 154.14, 144.52, 134.93, 127.89, 127.40, 126.60, 123.97, 121.59, 119.25,

105.27, 56.34, 22.19; HRMS: (m/z) calcd for CieHieNsCh [M+H]⁺: 334.1299, found 334.1305. 2-((7-Methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (8b) (ECH- 87): The title compound (8b) was obtained from the reaction of 3b with isatoic anhydride, as a white solid in 63% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 13.65 (s, 1H), 11.11 (s, 1H), 8.09 (d, J= 8.0 Hz, 1H), 8.05 (dd, J= 4.0 & 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.46 (d, J= 8.0 Hz, 1H), 7.32 (t, J= 8.0 Hz, 1H), 7.13 (d, J= 8.0 Hz, 1H), 7.09 (s, 1H), 3.99 (s, 3H), 2.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 170.46, 164.99, 155.94, 151.59, 148.63, 135.05, 128.30, 128.15, 126.62, 124.10, 119.26, 118.02, 115.99, 56.39, 21.85; HRMS: (m/z) calcd for CieHieNsCfe [M+H]⁺: 334.1299, found 334.1295.

2-((6,7-Dimethoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (8c or

KC47) (ECH-88): The title compound (8c) was obtained from the reaction of 3c with isatoic anhydride, as a white solid in 68% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 13.64 (brs, 1H), 11.01 (brs, 1H), 8.06 (d, J= 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.43-7.45 (m, 2H), 7.31 (t, J= 8.0 Hz, 1H), 7.13 (s, 1H), 4.03 (s, 3H), 3.96 (s, 3H), 2.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 168.09, 157.10, 154.57, 148.87, 146.98, 134.91, 126.59, 124.59, 123.84, 119.19,

115.81, 105.49, 104.69, 56.66, 56.49, 21.98; HRMS: (m/z) calcd for C19H18N5O3

[M+H]⁺: 364.1404, found 364.1406.

2-((5,6,7-Trimethoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (8d)

(ECH-89): The title compound (8d) was obtained from the reaction of 3d with isatoic anhydride, as a white solid in 73% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.55 (brs, 1H), 11.09 (brs, 1H), 8.05 (d, J= 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.45 (d, J= 8.0 Hz, 1H), 7.31 (t, J= 8.0 Hz, 1H), 6.97 (s, 1H), 4.04 (s, 3H), 4.00 (s, 3H), 3.84 (s, 3H), 2.93 (s, 3H); ¹³C NMR (150 MHz, DMSO- de), d (ppm): 168.77, 160.38, 155.04, 150.49, 148.46, 140.77, 134.91, 126.60, 124.02, 119.27, 111.64, 102.25, 100.08, 61.70, 61.24, 56.88, 26.41; HRMS: (m/z) calcd for C20H20N5O4 [M+H]⁺: 394.1510, found 394.1502.

2-((4-Methyl-7,8,9,10-tetrahydrobenzo[h]quinazolin-2-yl)amino)quinazolin-4(Li/)- one (8e) (ECH-90): The title compound (8e) was obtained from the reaction of 3f with isatoic anhydride, as a white solid in 67% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 13.83 (brs, 1H), 10.89 (brs, 1H), 8.06 (d, J= 8.0 Hz, 1H), 7.91 (d, J= 8.0 Hz, 1H), 7.73 (t, J= 8.0 Hz, 1H), 7.47 (d, J= 8.0 Hz, 1H),

7.32 (t, J= 8.0 Hz, 1H), 7.25 (d, J= 8.0 Hz, 1H), 3.14 (t, J= 8.0 Hz, 2H), 2.92 (d, J = 8.0 Hz, 2H), 2.86 (s, 3H), 1.95-1.97 (m, 2H), 1.85-1.87 (m, 2H); ¹³C NMR (100 MHz, DMSO-i¾), d (ppm): 171.70, 159.99, 154.93, 147.64, 144.67, 134.89, 131.57, 127.51, 126.54, 124.03, 123.05, 119.36, 118.94, 30.31, 24.35, 22.67, 22.48, 21.94; m/z (ESI MS): HRMS: (m/z) calcd for C21H20N5O [M+H]⁺: 358.1662, found 358.1653.

2-((4-Methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (8f) (ECH-91): The title compound (8f) was obtained from the reaction of 3g with isatoic anhydride, as a white solid in 63% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.70 (brs, 1H), 11.30 (brs, 1H), 8.24 (d, J= 8.0 Hz, 1H), 8.08 (d, J= 8.0 Hz, 1H), 7.97 (t, J= 8.0 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 7.74 (t, J= 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.48 (d, J= 8.0 Hz, 1H), 7.34 (s, 1H), 2.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-i¾), d (ppm): 172.14, 165.01, 155.49, 148.89, 135.65, 134.96, 126.70, 126.62, 126.30, 125.98, 124.17, 120.97, 119.39, 22.01; HRMS: (m/z) calcd for C17H14N5O

[M+H]⁺: 304.1193, found 304.1184.

2-((4-Methyl-6-morpholinoquinazolin-2-yl)amino)quinazolin-4(Li/)-one (8g or KC45) (ECH-92): The title compound (8g) was obtained from the reaction of 3h with isatoic anhydride, as a yellow solid in 60% yield by following the general procedure. 'H NMR (400 MHz, DMSO-^), d (ppm): 13.60 (brs, 1H), 10.81 (brs, 1H), 8.06 (d, J= 8.0 Hz, 1H), 7.80 (dd, J= 4.0 & 8.0 Hz, 1H), 7.71 (t, J= 8.0 Hz, 2H), 7.47 (d, J= 8.0 Hz, 1H), 7.29-7.32 (m, 2H), 3.80-3.81 (m, 4H), 3.29-3.30 (m, 4H), 2.87 (s, 3H); ¹³C NMR (100 MHz, DMSO-£¾), d (ppm): 169.95, 161.47, 153.60, 148.91, 143.52, 134.87,

127.17, 127.01, 126.59, 123.85, 121.74, 119.22, 107.10, 66.50, 48.99, 22.08; HRMS: (m/z) calcd for C21H22N6O2 [M+H]⁺: 389.1721, found 389.1716.

6-Chloro-2-((6-methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (9a) (ECH-93): The title compound (9a) was obtained from the reaction of 3a with 5- chloroisatoic anhydride, as a white solid in 64% yield by following the general procedure. ¾ NMR (600 MHz, DMSO-^), d (ppm): 13.74 (brs, 1H), 11.23 (brs, 1H), 7.96 (s, 1H), 7.71-7.73 (m, 2H), 7.59-7.62 (m, 1H), 7.43-7.48 (m, 2H), 3.93 (s, 3H),

2.90 (s, 3H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 170.36, 157.09, 153.89, 149.04, 144.45, 134.85, 128.02, 127.86, 127.36, 125.45, 121.63, 120.33, 105.26, 56.33, 22.11; HRMS: (m/z) calcd for C18H15CIN5O2 [M+H]⁺: 368.0909, found 368.0911.

6-Chloro-2-((7-methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (9b)

(ECH-94): The title compound (9b) was obtained from the reaction of 3b with 5- chloroisatoic anhydride, as a white solid in 67% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 13.77 (brs, 1H), 11.23 (brs, 1H), 8.09 (d, J= 8.0 Hz, 1H), 7.94 (d, J= 4.0 Hz, 1H), 7.72 (dd, J= 4.0 & 8.0 Hz, 1H), 7.44 (d, J= 8.0 Hz, 1H), 7.13 (dd, J= 4.0 & 8.0 Hz, 1H), 7.06 (s, 1H), 3.99 (s, 3H), 2.82 (s, 3H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 170.43, 160.37, 155.80, 151.72,

148.91, 134.94, 128.26, 128.18, 125.48, 120.46, 118.05, 116.17, 105.38, 56.42, 21.75; HRMS: (m/z) calcd for C18H15CIN5O2 [M+H]⁺: 368.0909, found 368.0911.

6-Chloro-2-((6,7-dimethoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one

(9c) (ECH-95): The title compound (9c) was obtained from the reaction of 3c with 5- chloroisatoic anhydride, as a white solid in 63% yield by following the general procedure. ¾ NMR (600 MHz, DMSO-^), d (ppm): 13.68 (brs, 1H), 10.80 (brs, 1H), 7.97 (brs, 1H), 7.70-7.71 (m, 1H), 7.47 (brs, 1H), 7.41 (s, 1H), 7.14 (brs, 1H), 3.94 (s, 3H), 2.89 (s, 3H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 168.10, 160.51, 157.23, 154.39, 149.04, 147.00, 134.88, 127.93, 125.47, 120.30, 115.93, 105.56, 104.79, 56.69, 56.55, 21.94; HRMS: (m/z) calcd for C19H15CIN5O3 [M+H]⁺: 398.1014, found

398.1011.

6-Chloro-2-((5,6,7-trimethoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)- one (9d) (ECH-96): The title compound (9d) was obtained from the reaction of 3d with 5-chloroisatoic anhydride, as a white solid in 71% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-^), d (ppm): 13.70 (brs, 1H), 11.21 (brs, 1H), 7.97 (d, J= 8.0 Hz, 1H), 7.74 (dd, J= 8.0 Hz, 1H), 7.45 (d, J= 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 4.04 (s, 1H), 4.00 (s, 1H), 3.85 (s, 3H), 2.97 (s, 3H), ¹³C NMR (150 MHz, DMSO-£¾), d (ppm): 168.85, 160.48, 154.86, 150.50, 148.42, 140.89, 134.95, 125.49, 120.41, 111.76, 102.30, 61.70, 61.25, 56.91, 26.37; HRMS: (m/z) calcd for C20H19CIN5O4 [M+H]⁺: 428.1120, found 428.1115. 6-Chloro-2-((4-methyl-7,8,9,10-tetrahydrobenzo[h]quinazolin-2- yl)amino)quinazolin-4(Li/)-one (9e) (ECH-97): The title compound (9e) was obtained from the reaction of 3f with 5-chloroisatoic anhydride, as a white solid in 67% yield by following the general procedure. ¾ NMR (600 MHz, DMSO-<¾), d (ppm): 14.08 (brs, 1H), 11.05 (brs, 1H), 7.98 (d, J= 8.0 Hz, 1H), 7.93 (d, J= 8.0 Hz, 1H), 7.71 (dd,

8.0 Hz, 1H), 7.47 (d, J= 8.0 Hz, 1H), 7.28 (d, J= 8.0 Hz, 1H), 3.14-3.15 (m, 2H), 2.93 (t, J = 8.0 Hz, 2H), 2.87 (s, 3H), 1.97 (t, J= 8.0 Hz, 2H), 1.88 (t, J= 8.0 Hz, 2H); ¹³C NMR (150 MHz, DMSO-i¾), d (ppm): 171.79, 158.45, 154.76, 147.59, 144.78, 134.92,

131.62, 128.09, 127.65, 125.42, 123.05, 120.47, 119.04, 30.33, 24.33, 22.66, 22.48, 21.92; HRMS: (m/z) calcd for C₂₁H₁₉CIN₅O [M+H]⁺: 392.1273, found 392.1273.

6-Chloro-2-((4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (9f) (ECH-98) : The title compound (9f) was obtained from the reaction of 3g with 5-chloroisatoic anhydride, as a white solid in 62% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 11.86 (brs, 1H), 10.03 (brs, 1H), 8.23 (d, J= 8.0 Hz, 1H), 8.01 (d, J= 8.0 Hz, 1H), 7.91-7.97 (m, 2H), 7.60 (t, J= 8.0 Hz, 1H), 7.11 (t, J =

8.0 Hz, 1H), 6.72 (d, J= 8.0 Hz, 1H), 2.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 172.09, 159.22, 154.78, 150.02, 149.19, 135.38, 132.08, 130.88, 127.01, 126.54, 126.34, 121.22, 119.37, 118.75, 117.72, 21.88; HRMS: (m/z) calcd for C17H13CIN5O [M+H]⁺: 338.0803, found 338.0809.

Synthesis procedure for synthesis of l-(4,6-Dimethylpyrimidin-2-yl)guanidine (10): The mixture of /V-(4,6-dimethylpyrimidin-2-yl)cyanamide (6) (8g, 0.054 mol) and ammonium chloride (12g, 0.23 mol) in phenol (25g, 0.27 mol) was heated at 125°C for 7 h. The reaction mixture was then poured into ice water and extracted with ethyl acetate, and washed with 2N sodium hydroxide solution. The organic layer was washed with brine, dried over anhydrous sodium sulphate and concentrated under reduced pressure to give the desired l-(4,6-dimethylpyrimidin-2-yl)guanidine in 68 % yield. ¾ NMR (400 MHz, DMSO-^e), d (ppm): 7.03 (brs, 4H), 6.44 (s, 1H), 2.21 (s, 6H); ¹³C NMR (100 MHz, DMSO-^e), d (ppm): 166.69, 166.01, 159.61, 110.31, 24.11; ESI MS (m/z): calculated for C₇H₁₂N₅ (M+H)⁺: 166.1073, found 166.2.

Synthesis of Bis(4,6-dimethylpyrimidin-2-yl)amine (11) (ECH-99): The mixture of 1- (4,6-dimethylpyrimidin-2-yl)guanidine (198 mg 1.2 mmol) and acetylacetone (242mg, 2.42 mmol) in acetic acid (0.5 mL) was heated at 125°C for 12 h. The progress of the reaction was monitored with TLC. Upon completion, the reaction mixture was allowed to cool to room temperature and was basified with 5 mL of ammonia (28%) in ice cold water to give a precipitate which were filtered and purified through column

chromatography over silica using 0.5-1.5% of MeOH in DCM as solvent system to give the desired product in 64% yield. ¾ NMR (400 MHz, CDCL), d (ppm): 7.87 (brs, 1H), 6.64 (s, 2H), 2.43 (s, 12H); ¹³C NMR (100 MHz, CDCL), d (ppm): 167.98, 158.33, 113.91, 24.07; HRMS: (m/z) calcd for C_I2H_I6N₅ [M+H]⁺: 230.1400, found 230.1402.

General procedure for synthesis of 2-((4,6-dimethylpyrimidin-2- yl)amino)quinazolin-4(Li/)-one derivatives 12-17: To the mixture of l-(4,6- dimethylpyrimidin-2-yl)guanidine (10) (150 mg, 0.91 mmol) and isatoic anhydride (176 mg, 1.08 mmol) in anhydrous DMF (1.5 mL), diisopropyl ethylamine (188 pL, 1.08 mmol) was added and reaction mixture was heated at 100°C for 12 h. Progress of the reaction mixture was monitored using TLC. Upon completion, the reaction mixture was concentrated under reduced pressure and purified through column chromatography over silica using MeOH (0-2% ) and triethylamine (0.2%) in DCM to give the pure 2-((4,6- dimethylpyrimidin-2-yl)amino)quinazolin-4(U/)-one derivatives in 43-57 % yield.

2-((4,6-Dimethylpyrimidin-2-yl)amino)quinazolin-4(LiF)-one (12) (ECH-100): The title compound (12) was obtained from the reaction of 10 with isatoic anhydride, as a white solid in 57% yield by following the general procedure. 'H NMR (400 MHz, CDCL), d (ppm): 13.27 (brs, 1H), 9.20 (brs, 1H), 8.23 (dd, J= 4.0 & 8.0 Hz, 1H), 7.63 (t, J= 8.0 Hz, 1H), 7.50 (dd, J= 4.0 & 8.0 Hz, 1H), 7.29 (t, J= 8.0 Hz, 1H), 6.70 (s, 1H), 2.46 (s, 6H); ¹³C NMR (100 MHz, CDCL), d (ppm): 168.25, 161.94, 157.89, 149.74, 147.78, 134.35, 126.71, 125.76, 123.99, 119.39, 114.45, 23.85; HRMS: (m/z) calcd for C14H14N5O [M+H]⁺: 268.1193, found 268.1195.

6-Chloro-2-((4,6-dimethylpyrimidin-2-yl)amino)quinazolin-4(LiF)-one (13) (ECH- 101): The title compound (13) was obtained from the reaction of 10 with 5-chloroisatoic anhydride, as a white solid in 55% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.47 (brs, 1H), 10.80 (brs, 1H), 7.95 (d, J= 4.0 & 8.0 Hz, 1H), 7.69 (dd, J= 4.0 & 8.0 Hz, 1H), 7.44 (dd, J= 8.0 Hz, 1H), 6.94 (s, 1H), 2.43 (s, 6H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 168.12, 160.41, 158.43, 148.93, 134.91, 128.11, 127.81, 125.51, 125.42, 120.33, 114.70, 23.80; HRMS: (m/z) cal cd for C₁₄H₁₃CIN₅O [M+H]⁺: 302.0803, found 302.0807.

2-((4,6-Dimethylpyrimidin-2-yl)amino)-8-methoxyquinazolin-4(Lif)-one (14) (ECH-

102): The title compound (14) was obtained from the reaction of 10 with 3- methoxyisatoic anhydride (obtained from the reaction of 2-amino-3-methoxybenzoic acid with triphosgene) (Shikhaliev et al., 2002), as a white solid in 47% yield by following the general procedure. ¹H NMR (400 MHz, DMSO-<¾), d (ppm): 13.95 (brs, 1H), 10.87 (brs, 1H), 7.59 (d, J= 8.0 Hz, 1H), 7.25-7.32 (m, 2H), 6.97 (s, 1H), 3.99 (s, 3H), 2.46 (s, 6H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 167.94, 160.05, 158.95, 152.52, 147.55, 129.31, 124.16, 119.69, 118.11, 114.58, 56.72, 23.81; HRMS: (m/z) calcd for C15H16N5O2 [M+H]⁺: 298.1299, found 298.1292.

2-((4,6-Dimethylpyrimidin-2-yl)amino)-7-methoxyquinazolin-4(Lif)-one (15) (ECH-

103): The title compound (15) was obtained from the reaction of 10 with 4- methoxyisatoic anhydride (obtained from reaction 2-amino-4-methoxybenzoic acid with triphosgene) (Shikhaliev et al., 2002), as a white solid in 50% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-_<¾), d (ppm): 13.19 (brs, 1H), 10.71 (brs, 1H), 7.94 (d, J= 8.0 Hz, 1H), 6.94 (s, 1H), 6.89 (dd, J= 8.0 Hz, 1H), 6.84 (s, 1H), 3.88 (s, 3H), 2.43 (s, 6H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 168.08, 164.73, 160.77, 158.42, 152.67, 148.96, 128.17, 114.56, 113.62, 112.60, 106.85, 56.98, 23.87; HRMS: (m/z) calcd for C15H16N5O2 [M+H]⁺: 298.1299, found 298.1299.

6-Bromo-2-((4,6-dimethylpyrimidin-2-yl)amino)quinazolin-4(Lif)-one (16) (ECH-

104): The title compound (16) was obtained from the reaction of 10 with 5-bromoisatoic anhydride as a white solid in 45% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 13.50 (brs, 1H), 10.91 (brs, 1H), 8.10 (d, J= 8.0 Hz, 1H), 7.83 (d, J= 8.0 Hz, 1H), 7.39 (d, J= 8.0 Hz, 1H), 6.97 (s, 1H), 2.44 (s, 6H); ¹³C NMR (150 MHz, DMSO-^), d (ppm): 168.15, 160.47, 158.42, 149.12, 137.64, 128.56, 127.76, 120.80, 115.88, 114.75, 23.82; HRMS: (m/z) calcd for Ci₄Hi₃BrN₅0 [M+H]⁺: 346.0298, found 346.0297.

2-((4,6-Dimethylpyrimidin-2-yl)amino)-6,7-difluoroquinazolin-4(Lif)-one (17)

(ECH-105): The title compound (17) was obtained from the reaction of 10 with 4,5- difluoroisatoic anhydride (obtained from reaction 2-amino-4,5-difluorobenzoic acid with triphosgene) (Shikhaliev et al., 2002), as a white solid in 43% yield by following the general procedure.¹H NMR (600 MHz, DMSO-_<¾), d (ppm): 13.50 (brs, 1H), 10.85 (brs, 1H), 7.89-7.92 (m, 1H), 7.37-7.40 (m, 1H), 6.97 (s, 1H), 2.45 (s, 6H); ¹³C NMR (150 MHz, DMSO-i¾), d (ppm): 168.16, 160.13, 158.38, 155.49 (d J_C-F= eo Hz), 153.81

(d C-F= 60 Hz), 149.33, 148.01(d, C-F= 60 Hz), 146.39 (d, C-F= 54 Hz), 115.94(d, C-F= 24 Hz), 114.77, 113.89 39 (d, J_C-F= _IS H_z), 23.80; HRMS: (m/z) calcd for CwHnFiNaNsO [M+Na]⁺: 326.0823, found 326.0826. Based on the compounds 4f and 8g, a series of new derivatives with different substituents as shown in Scheme-5 were synthesized.

Scheme 5. Reagents and conditions: i. dry acetone, h, /-butyl catechol, MgS0₄, reflux 16-18 h, (33-47%); ii. 2-cynogunidine, 2M HC1, 100°C, 0.5 h, (29-54 %); iii. acetylacetone, acetic acid, reflux, 12 h, (31%); iv. Isatoic anhydride, DIPEA, 100°C, 12 h, (31-58%); v. acetic acid-HBr (7:3), reflux, 12 h, (66%).

General procedure for the synthesis of 2,2,4-trimethyl-l,2-dihydroquinoline derivatives 2(i-r): The title compounds 2(i-r) shown in Scheme-5 were synthesized by following the same procedure used for the synthesis of 2(a-k). The purification of 2(i-r) was performed through column chromatography over silica with 0-10% ethyl acetate in heptane as solvent system to give 2(i-r) in 33-47% yield. General procedure for the synthesis of l-(4-methylquinazolin-2-yl)guanidine derivatives (3i-r): The title compounds (3i-r) shown in Scheme-5 were synthesized by following the same procedure used for synthesis of 3(a-k). The purification of 3(i-r) was performed through crystallization water (80%) methanol (18%) and saturated ammonium hydroxide (2%) mixture to give the desired compounds in 29-54% yield.

Procedure for the synthesis of /V-(4,6-dimethylpyrimidin-2-yl)-4-methyl-8,9- dihydro-7//-cyclopenta[/i]quinazolin-2-amine (4i or KC243):

The title compound was obtained by refluxing the reaction mixture of l-(4-methyl-8,9- dihydro-7//-cyclopenta[h]quinazolin-2-yl (guanidine (3i) (94 mg, 0.39 mmol) and acetylacetone (80pL, 0.78 mmol) in acetic acid (0.5 mL) for 12 h. Progress of the reaction was monitored using TLC. Upon completion, the reaction mixture was basified (pH ~ 8-9) and extracted with 2X50 mL of DCM. The combined organic layer were dried over sodium sulphate, filtered, concentrated, and purified through column

chromatography over silica gel with MeOH (0-2%) and triethylamine (0.2%) in DCM as solvent system to give the desired /V-(4,6-dimethylpyrimidin-2-yl)-4-methyl-8,9- dihydro-7H-cyclopenta[h]quinazolin-2-amine (4i) in 31% yield as a light brown solid. ¾ NMR (400 MHz, CDCh), d (ppm): 7.80 (d, J= 8.0 Hz, 1H), 7.31 (d, J= 8.0 Hz,

1H), 6.70 (s, 1H), 3.38 (t, J= 8.0 Hz, 2H), 3.11 (t, J= 8.0 Hz, 2H), 2.87 (s, 3H), 2.48 (s, 6H), 2.20-2.27 (m, 2H); ¹³C NMR (100 MHz, CDCh), d (ppm): 168.79, 167.25, 157.98, 153.48, 149.69, 142.19, 133.16, 125.92, 121.01, 118.55, 113.10, 29.88, 23.71, 23.56, 22.28, 21.23; m/z (ESI MS): calculated for C_I8H_2ON₅ (M+H)⁺: 306.38; obtained 306.3.

General procedure for the synthesis of 2-((4-methylquinazolin-2- yl)amino)quinazolin-4(Li/)-one 8(h-q): The title compounds 3(h-q) shown in Scheme- 5 were synthesized by following same procedure used for synthesis of 8(a-g). The purification of 8(h-p) was performed through crystallization in DMF which gave the desired compounds in 21-47% yield. 2-((4-methyl-6-(piperidin-l-yl)quinazolin-2-yl)amino)quinazolin-4(Lif)-one (8h or KC240):

The title compound (8h) was obtained from the reaction of 1 -(4-methyl -6-(piperi din- 1- yl)quinazolin-2-yl)guanidine (3j) with isatoic anhydride, as a light yellow solid in 44% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.68 (brs, 1H), 11.00 (brs, 1H), 8.04 (d, = 8.0 Hz, 1H), 7.79 (dd, J= 4.0 & 8.0 Hz, 1H), 7.71 (t, J= 8.0 Hz, 1H), 7.62 (d, J= 8.0 Hz, 1H), 7.44 (d, J= 8.0 Hz, 1H), 7.30 ((t, J= 8.0 Hz, 1H), 7.23 (s, 1H), 3.26-3.28 (m, 4H), 2.84 (s, 3H), 1.64-1.68 (m, 4H), 1.57- 1.58 (m, 2H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 169.71, 161.37, 153.60,

150.32, 150.66, 149.41, 148.66, 134.98, 128.21, 126.75, 126.59, 125.62, 123.79,

121.76, 119.13, 107.03, 49.89, 25.63, 24.22, 22.14; m/z (ESI MS): calcd for C₂₂H₂₃N₆O (M+H)⁺: 387.19; obtained 387.2. 2-((4-methyl-7-morpholinoquinazolin-2-yl)amino)quinazolin-4(Lif)-one (8i or

KC234):

The title compound (8i) was obtained from the reaction of 1 -(4-methyl -7- morpholinoquinazolin-2-yl)guanidine (3k) with isatoic anhydride, as a light yellow solid in 47% yield by following the general procedure. 'H NMR (400 MHz, DMSO-<¾), d (ppm): 13.81 (brs, 1H), 10.96 (brs, 1H), 8.05 (d, J= 8.0 Hz, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.72 (t, J= 8.0 Hz, 1H), 7.45 (d, J= 4.0 Hz, 1H), 7.29-7.35 (m, 2H), 6.87 (s, 1H), 3.77-3.80 (m, 4H), 3.42-3.45 (m, 4H), 2.77 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 169.30, 163.55, 155.77, 151.32, 134.89, 133.37, 132.02, 127.60, 126.60,

123.91, 119.32, 116.02, 114.42, 111.36, 105.11, 99.99, 66.35, 47.45, 21.44 m/z (ESI MS): calcd for CiiHziNeOi (M+H)⁺: 389.17; obtained 389.3. 2-((6-(Diethylamino)-4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (8j or KC246):

The title compound (8j) was obtained from the reaction of l-(6-(diethylamino)-4- methylquinazolin-2-yl)guanidine (31) with isatoic anhydride, as a green-yellow solid in 43% yield by following the general procedure. 'H NMR (400 MHz, DMSO-<¾), d (ppm): 13.73 (brs, 1H), 10.93 (brs, 1H), 8.04 (d, J= 8.0 Hz, 1H), 7.71 (t, J= 8.0 Hz, 1H), 7.63 (d, J= 8.0 Hz, 1H), 7.57 (dd, J= 4.0 & 8.0 Hz, 1H), 7.43 (d, J= 8.0 Hz, 1H), 7.29 ((t, J= 8.0 Hz, 1H), 6.94 (s, 1H), 3.48 (q, J= 8.0 Hz, 4H), 2.82 (s, 3H), 1.16 ((t, J = 8.0 Hz, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 168.80, 161.42, 152.28,

150.76, 148.73, 145.55, 140.84, 134.99, 127.12, 126.58, 125.57, 124.31, 123.65,

122.44, 119.06, 102.24, 44.34, 22.13, 12.72; m/z (ESI MS): calcd for CiiHisNeO (M+H)⁺: 375.19; obtained 375.2. 2-((6-Bromo-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (8k or KC235):

The title compound (8k) was obtained from the reaction of l-(6-bromo-4- methylquinazolin-2-yl)guanidine (3m) with isatoic anhydride, as a light yellow solid in 26% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.43 (brs, 1H), 11.34 (brs, 1H), 8.41 (d, J= 4.0 Hz, 1H), 8.02-8.06 (m, 2H), 7.71-7.73 (m, 2H), 7.47 (d, J= 8.0 Hz, 1H), 7.33 ((t, J= 8.0 Hz, 1H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 171.77, 161.30, 155.68, 148.25, 147.61, 138.47, 135.14, 135.06, 128.93, 128.51, 126.62, 124.30, 122.08, 119.30, 118.06, 99.98, 22.21; m/z (ESI MS): calcd for Ci₇Hi₃BrN₅0 (M+H)⁺: 382.03; obtained 382.1.

2-((6-Chloro-4-methylquinazolin-2-yl)amino)quinazolin-4(Lif)-one (81 or KC250):

The title compound (81) was obtained from the reaction of l-(6-Chloro-4- methylquinazolin-2-yl)guanidine (3n) with isatoic anhydride, as a grey solid in 40% yield by following the general procedure. ¹H NMR (400 MHz, DMSO-<¾), d (ppm): 13.42 (brs, 1H), 11.37 (brs, 1H), 8.28 (d, J= 4.0 Hz, 1H), 8.05 (d, J= 8.0 Hz, 1H), 7.93 (d, J= 8.0 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 7.73 ((t, J= 8.0 Hz, 1H), 7.48 (d, J= 8.0 Hz, 1H), 7.33 (t, J= 8.0 Hz, 1H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 171.81, 163.53, 155.66, 148.38, 147.41, 140.08, 135.89, 135.03, 129.81, 128.41, 126.60, 125.78, 124.29, 121.53, 119.29, 112.38, 22.20; m/z (ESI MS): calcd for C17H13CIN5O (M+H)⁺: 338.08; obtained 338.1.

2-((6-Fluoro-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (8m or KC253):

The title compound (8m) was obtained from the reaction of l-(6-fluoro-4- methylquinazolin-2-yl)guanidine (3o) with isatoic anhydride, as a light yellow solid in 39% yield by following the general procedure. . ¾ NMR (400 MHz, DMSO-i¾), d (ppm): 13.47 (brs, 1H), 11.29 (brs, 1H), 8.01-8.06 (m, 2H), 7.85-7.86 (m, 2H), 7.73 (t, J = 8.0 Hz, 1H), 7.47 (d, J= 8.0 Hz, 1H), 7.32 (t, J= 8.0 Hz, 1H), 2.88 (s, 3H); (ESI MS): calcd for C₁₇H₁₃FN₅O (M+H)⁺: 322.31; obtained 322.1.

2-((8-Methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(LiF)-one (8n or

KC261):

The title compound (8n) was obtained from the reaction of l-(8-m ethoxy -4- methylquinazolin-2-yl)guanidine (3p) with isatoic anhydride, as a grey solid in 38% yield by following the general procedure. ¾ NMR (400 MHz, DMSO-6/7), d (ppm): 13.99 (brs, 1H), 11.06 (brs, 1H), 8.06 (d, J= 8.0Hz, 1H), 7.70-7.75 (m, 2H), 7.42-7.49 (m, 3H), 7.33 (t, J= 8.0 Hz, 1H), 4.07 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-£¾), d (ppm): 171.97, 162.28, 153.37, 150.36, 149.42, 140.34, 134.80, 126.62, 125.77, 124.04, 121.47, 119.49, 118.83, 117.54, 114.15, 99.99, 56.81, 22.33; (ESI MS): calcd for CisHieNsCfe (M+H)⁺: 334.13; obtained 334.2

2-((6-(4-(2-Aminobenzoyl)piperazin-l-yl)-4-methylquinazolin-2- yl)amino)quinazolin-4(Li/)-one (8o or KC281):

The title compound (8o) was obtained from the reaction l-(4-methyl-6-(piperazin-l- yl)quinazolin-2-yl)guanidine (3q) with isatoic anhydride, as a yellow colored solid in 21% yield by following the general procedure. 'H NMR (400 MHz, DMSO-i¾), d (ppm): 13.68 (brs, 1H), 11.07 (brs, 1H), 8.05 (d, J= 8.0Hz, 1H), 7.84 (d, J= 8.0Hz,

1H), 7.68-7.74 (m, 2H), 7.44 (d, J= 8.0 Hz, 1H), 7.28-7.34 (m, 2H), 7.12 (t, J= 8.0 Hz, 1H), 7.06 (d, J= 8.0 Hz, 1H), 6.73 (d, J= 8.0 Hz, 1H), 6.59 (t, J= 8.0 Hz, 1H), 5.22 (brs, 2H), 3.66 (brs, 6H), 3.37-3.38 (m, 4H), 2.86 (s, 3H); ¹³C NMR (100 MHz, DMSO- de), d (ppm): 170.11, 169.15, 162.76, 161.37, 153.58, 150.66, 148.59, 146.25, 143.42, 135.04, 130.57, 128.28, 128.03, 126.97, 126.59, 125.68, 123.86, 121.67, 119.57,

119.17, 115.99, 115.96, 107.84, 49.01, 36.25, 31.24, 22.17; (ESI MS): calcd for C28H27N8O2 (M+H)⁺: 507.22; obtained 507.3.

2-((6-Amino-4-methylquinazolin-2-yl)amino)quinazolin-4(Li/)-one (8q or KC298):

To obtain the title compound (8q), firstly benzyl (4-methyl -2 -((4-oxo- 1,4- dihydroquinazolin-2-yl)amino)quinazolin-6-yl)carbamate (8p) was prepared from the reaction of benzyl (2-guanidino-4-methylquinazolin-6-yl)carbamate (3r) with isatoic anhydride, by following the general procedure to give 8p in 58% yield. Acidic hydrolysis of benzyl (4-methyl -2-((4-oxo-l,4-dihydroquinazolin-2- yl)amino)quinazolin-6-yl)carbamate (8p) was performed in 0.25 mL of acetic acid-HBr (7:3) mixture under refluxing conditions for 12 h. The reaction mixture was then concentrated under reduced pressure, poured into crushed ice, and basified (pH ~ 8-9) to give a brown solid. The brown solid was purified through column chromatography on silica gel by eluting with methanol (0-2% ) and triethylamine (0.2%) in DCM to give the desired compound (8q) in 66% yield. ¾NMR (400 MHz, DMSO-<¾), d (ppm): 13.76 (brs, 1H), 10.91 (brs, 1H), 8.05 (d, J= 4.0Hz, 1H), 7.71 (t, J= 8.0 Hz, 1H), 7.56 (d, J= 8.0 Hz, 1H), 7.43 (d, J= 8.0 Hz, 1H), ), 7.37 (d, J= 8.0 Hz, 1H), 7.29 (t, J= 8.0 Hz, 1H), 7.05 (s, 1H), 5.72 (s, 2H), 2.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-^), d (ppm): 168.34, 161.43, 152.20, 150.80, 148.79, 147.21, 141.46, 135.01, 127.00, 126.88, 126.59, 125.58, 123.65, 112.44, 119.06, 103.68, 22.01; (ESI MS): calcd for CnHisNeO (M+H)⁺: 319.13; obtained 319.2

EXAMPLE 2: G4 stabilization in vitro

The G4 stabilization effect of the analogues was determined by measuring the progression of Taq DNA polymerase on DNA templates carrying G4 structures with different topologies and one non-G4 control DNA template (Jamroskovic et al., 2016) (Han et al., 1999). Several selective G4-stabilizing compounds and structure-function relationships were identified (Figure 7). For subsequent experiments, compounds 4f and 8g (Figure la) were selected, since these were the most effective in stabilizing the different G4 topologies without affecting the non-G4 DNA (Figure 7). Dose-dependent studies of these two compounds showed an up to 10-fold improvement in G4-associated inhibition of DNA polymerization compared to the original hit 5b, suggesting that these two compounds efficiently stabilize G4 structures (Figures lb and c, Figure 8a). The topologies of the G4 structures had slightly different impacts on the stabilization ability of the compounds, and 5b and 8g had stronger preferences for parallel DNA structures than a hybrid DNA structure, while 4f stabilized the ribosomal parallel and hybrid DNA structures equally well, and showed very strong preference for the well-characterized parallel c-MYC Pu24T G4 structure (Figure lb and c, Figure 8a).

Surface plasmon resonance (SPR) was used to confirm that both compounds bind to G4 structures (Figures Id and e and Figures 8b-h). The dissociation constant (K_d) for 4f for c-MYC Pu24T G4 DNA structure was estimated by both SPR and microscale thermophoresis (MST) to be -180 nM (Figures ld-g) (the physicochemical properties of 8g prevented exact K_d estimations). Moreover, the compounds were selective for G4 DNA over single-stranded DNA (ssDNA) because the affinity of 4f and 8g measured by SPR and/or fluorescence titrations for the ssDNA control oligonucleotide was negligible (Figures 8d, 8h, and 15).

In agreement with the SPR, MST, and/or fluorescence titration results, the NMR data showed that both 4f and 8g bound to the c-MYC Pu24T G4 structure (Figures ld-g, 9a and b, and 15b-f). However, the chemical shift changes could not be quantified for 8g because line broadening of the imino peaks was observed instead of a new set of peaks (data not shown), which suggest multiple binding modes or fast on-off rates. By mapping the peak shift changes induced by 4f to the c-MY C Pu24T G4 NMR structure, we found that 4f strongly affected two of the guanines on one side of the top G-tetrad (G-4 and G-8) (Figure lh and Figures 9a and b) and guanine G-5 in the second G-tetrad located below G-4 and G-8 (Figure lh). The strong binding to one side of the top G- tetrad could potentially be explained by binding interactions with the 5' DNA sequence flanking the G4 or by a more atypical intercalative binding mode.

EXAMPLE 3 : Molecular dynamics (MD) simulations

To investigate these potential binding modes, molecular dynamics (MD) simulations were performed based on the NMR results. When 4f was modeled on the top of the first G-tetrad, it mostly interacted with G-4 and G-8 in the first G-tetrad but also with G-17, although no chemical shift changes were observed for G-17 in the NMR experiments (Figure 9c-d). When the compound was intercalated between the first and the second G- tetrad it also generated a stable structure where 4f mostly interacted with G-4 and G-8, leaving G-17 largely unaffected in accordance with the NMR results (Figure li-k). The MD-predicted affinity for the top-binding mode was weaker than for the intercalating conformation. The intercalative binding mode would explain 4f s high affinity despite being neutral and having a low molecular weight which is rare for compounds that target large and flat binding surfaces such as end stacking with G4 structures.

Nevertheless, the intercalative binding mode is unusual for G4 stabilizing compounds and even though the data match better for this binding mode, the most commonly described end-stacking binding mode cannot be excluded without further structural elucidations. EXAMPLE 4: Cell imaging

It was investigated whether the compounds are able to enter into cultured human cells. Based on the fluorescence properties of 8g (Figure 10a), imaging of live HeLa cells by 2 -photon excitation microscopy (Figure 10b) and confocal laser scanning microscopy (CLSM) (data not shown) showed the accumulation of 8g in the nucleolar G4-rich regions already at 10 min after the start of the treatment. Moreover, it was confirmed 4f, 5b, and 8g uptake by Caco-2 cell permeability experiments (Table 1). Together these data show that all three compounds are able to enter into human cells.

EXAMPLE 5: Cell toxicity

Because human cancer cell lines, and HeLa cells in particular, have increased amounts of G4 DNA structures compared to non-cancerous cells like human primary fibroblasts (HPFs) (Biffi et ak, 2013), the effect of 5b, 4f, and 8g on these two cell types were compared. Increasing concentrations of all three compounds were toxic to both HPFs and HeLa cells (4f and 8g were both more toxic compared to 5b) (Figure 2a and b). Notably, while the dose response of 5b was the same in the two cell types, 4f was slightly more toxic to HeLa cells and 8g had a significantly stronger effect on HeLa cells compared to HPFs (at 2.5-7.5 mM) (Figures lOc-e). The largest difference in cell survival was observed when HPFs (90.4% viable cells) and HeLa cells (8.6% viable cells) were treated with 2.5 mM of 8g (Figure lOe), showing that HeLa cells are about 10-fold more sensitive to 8g than HPFs.

One explanation for the observed cell viability effects might be perturbed DNA replication (Xu et ak, 2017). To examine the effect of 8g on DNA replication, DNA fiber analysis in HeLa cells (Figures 2c and d) was performed. The mean DNA replication tract length was significantly shorter in 8g-treated cells compared to mock- treated cells (p = 8.1 x 10^-18) (Figure 2e), suggesting that 8g affects the DNA

replication speed. Decreased DNA replication speed could potentially be a sign of DNA damage accumulation, which can be detected by phosphorylated histone H2A.X

(gH2A.C) protein levels (Kuo and Yang, 2008). HeLa cells were treated with increasing concentrations of 5b, 4f, and 8g based on the effects detected in the cell viability assay (Figure 2a), and an increased gH2A.C signal compared to the mock-treated cells (Figures 2f and g) was found. The upstream phosphorylation of ATM, the major kinase involved in the phosphorylation of H2A.X (Burma et ak, 2001) was also analyzed. The individual presence of all three compounds increased ATM phosphorylation levels, confirming that they all induce a DNA damage checkpoint response (Figures 2f and g). Surprisingly, 8g showed a differential dose response for ATM and H2A.X

phosphorylation (Figures 2f and g). At 5 mM 8g, the gH2A.C levels were increased, but the ATM phosphorylation levels were unchanged, whereas at higher compound concentrations both ATM and H2A.X were phosphorylated (Figures 2f and g). These data suggest that cells treated with 5 mM 8g undergo ATM-independent

phosphorylation of H2A.X, while at higher concentrations the compound causes increased gH2A.C through the ATM-dependent pathway. It has been reported that in cells treated with ionizing radiation a lower radiation dose leads to a strong decrease in the cell survival rate as a result of ATM-independent H2A.X phosphorylation. On the other hand, high radiation doses lead to phosphorylation of ATM and consequently DNA repair activation and thus have a less pronounced effect on cell survival (Collis et ak, 2004). A similar mechanism for 8g was confirmed by measuring apoptosis with increasing compound concentrations. At 5 mM, the proportion of apoptotic cells was about two-fold higher (23.1%) compared to cells treated with either 10 mM or 20 mM 8g (10.7% and 13%, respectively) (Figures 2h and 1 la). This finding explains the inverted cell viability dose response with 8g (Figure 2a).

It was found that HeLa cells treated with 4f had an approximately four-fold increase in the formation of internuclear chromatin bridges (ICBs) (Figures l i b and c), a hallmark of telomere instability (Veldman et ak, 2004). Similar to 4f, the G4-stabilizing compounds, CX-5461 and CX3543, also induce replication defects, DNA damage, and telomere instability, all important properties for DNA-targeting cancer drugs (Xu et ak, 2017) In fact, the highly aggressive triple negative breast cancer cell lines, MDA-MB- 231 and MDA-MB-436, are among the most sensitive breast cancer cell lines towards CX-5461 (Xu et ak, 2017). Furthermore, MDA-MB-436 cells have a BRCAl mutation that results in loss of nuclear BRCA1 protein expression (Elstrodt et al, 2006). These cell lines were treated with 4f to examine if MDA-MB-231 and MDA-MB-436 are also more sensitive to 4f than healthy epithelial cell lines derived from benign proliferative breast tissue. Indeed at 7 mM, a concentration that was not toxic for the control breast cell lines, reduced viability of both the MDA-MB-231 and MDA-MB-436 tumor cell lines, 60 and 55%, respectively, was found (Figure l ie). In addition, and similar to CX- 5461, both cell lines were more sensitive to 4f compared to the non-invasive and less aggressive breast cancer cell line MCF-7 which is BRCA1+/+ and do not contain known mutations in DNA damage repair genes (Figure l ie). These data demonstrate that triple negative breast cancer cell lines are more sensitive to 4f-treatment, than cell lines derived from control breast tissue, suggesting that 4f, similar to CX5461 that is in clinical trial phase I (Xu et al., 2017), may be a good drug candidate in treating triple negative breast cancer and target tumor cells that are deficient in DNA damage repair pathways. Similarly to 4f, the breast cancer cells MCF-7, MDA-MB-231, and MDA- MB-436 are more sensitive to 8g compared to the cell line derived from benign proliferative breast tissue, MCF-lOa.

EXAMPLE 6: G4 DNA stabilization in cells

To determine if 4f and 8g stabilize G4 DNA structures in human cell culture, the anti- G4 DNA antibody BG4 (Biffi et al., 2013) was used for immunofluorescence microscopy to visualize and quantify G4 DNA structures in HeLa cells. At the compound concentrations that resulted in a DNA damage response (Figures 2f and g), it was found that the number of BG4 foci per cell nucleus increased significantly in the treated cells compared to mock-treated cells (4f p = 5.5 ^c KG¹³ and 8g p = 1.06 ^c KG⁶) (Figures 3a-c). The increased number of BG4 foci could already be detected after 1 h treatment with 20 mM 8g (Figure 1 Id). In contrast, cells treated with 5 mM 8g, a concentration that caused phosphorylation of H2A.X without ATM activation, did not show an increase in the number of BG4-positive foci/cell, suggesting that the ATM- independent phosphorylation of H2A.X is not dependent on G4 stabilization (Figure l id). Together, these data support the hypothesis that 8g and 4f are able to stabilize G4 DNA structures in cells and that this induces replication stress and DNA damage and thus reduces cell viability. However, 8g affected the viability more strongly compared to 4f, and this cannot be explained only by G4 DNA stabilization because 4f is at least as effective as 8g in stabilizing G4 DNA structures both in vitro and in human cells (Figures lb, lc, 3, 7a and b). EXAMPLE 7: pSTAT3 inhibition

It has previously been reported (Jamroskovic et al., 2016) that a compound with structural similarities to compound 5b had been identified in a screen for compounds that selectively inhibit phosphorylation of STAT3 at tyrosine 705 (pSTAT3) (LaPorte et al., 2014). To test whether the new compounds affect pSTAT3 levels in human cells, treated HeLa cells were treated with 4f, 5b, or 8g, which indeed resulted in a reduction of the pSTAT3 protein levels for all three compounds (Figure 4a). In 4f-treated cells, pSTAT3 reduction occurred at 50 mM (Figures 4a and b), a concentration at which G4 stabilization was also increased (Figure 3c). In contrast, in 8g-treated cells pSTAT3 was inhibited already at 5 mM (Figures 4a and b), a concentration at which no increase in the number of BG4-positive foci (Figure l id) was detected. Together these data suggest that 4f and 8g act on both G4 structures and pSTAT3, although 8g-dependent pSTAT3 inhibition occurs at lower concentrations than the G4 structure stabilization, resulting in the activation of two different processes that ultimately lead to cell death (Figure 2h).

At the highest concentration tested for each of the compounds, total STAT3 levels were also affected (Figure 4a). However, the reduction of pSTAT3 occurred at lower compound concentrations, and a dose-dependent reduction of the pSTAT3/STAT3 ratio was observed (Figure 4b), indicating that the reduced pSTAT3 levels were not dependent on the total STAT3 protein levels. Because STAT3 levels are positively autoregulated (Narimatsu et al., 2001), STAT3 downregulation might represent a consequence of pSTAT3 inhibition. Alternatively, the gene encoding STAT3 contains a G4 motif in its promoter region (Lin et al., 2016), and a reduction in STAT3 levels might therefore be due to the stabilization of the G4 structure in the STAT3 promoter region. A direct interaction between 4f and 8g with STAT3 protein was confirmed by SPR analysis (4f K_d = 45 mM, 8g K_d = 15.5 mM) (Figures 4c-e), suggesting that 4f and 8g bind to the STAT3 protein and might therefore directly interfere with STAT3 phosphorylation.

STAT1, another member of the STAT family of proteins that have anti -proliferative and pro-apoptotic functions (Avalle et al., 2012), shares around 50% amino acid sequence homology with STAT3 (Szelag et al., 2015). Importantly, none of the compounds tested here affected the total or phosphorylated levels of STAT1 (Figure 4a, b), indicating that the compounds selectively inhibit STAT3 over STATE Therefore, 4f and 8g not only stabilize G4 structures, but also selectively inhibit the STAT3 -mediated pathway, which is an important pathway in cancer therapeutics.

EXAMPLE 8: Compound 8g localizes into the nucleus in S. pombe cells and perturbs replication fork progression

Although the JAK/STAT signaling pathway is essential for multicellular organisms, it is not present in unicellular organisms such as the fission yeast Schizosaccharomyces pombe (Miller, 2012; Tong et al., 2017). However, the positions of many G4 structures are conserved between S. pombe and multicellular organisms (Sabouri et al., 2014; Wallgren et al., 2016), and unresolved G4 structures result in fork pausing and DNA damage (McDonald et al., 2016; Sabouri et al., 2014), indicating that G4 structures also form in S. pombe. Therefore, to confirm that the effects of 4f and 8g on HeLa cells is a consequence also of G4 stabilization and not merely a result of the pSTAT3 inhibition, the effect of 8g was tested in S. pombe. For this study, an S. pombe mutant strain was employed in which the multi-drug resistant response pathway has been deleted, as wild- type S. pombe cells are multi-drug resistant due to very efficient drug efflux pumps (Arita et al., 2011; Kawashima et al., 2012b). It was found that 8g localized into the nucleus (Figure 5a) and affected the growth of asynchronous S. pombe cells (Figure 5b). 4f did not affect cell growth, probably due to a limited cell uptake in S. pombe (Figure 5b). Cells treated with 8-12 mM of 8g showed a four-fold decrease in the number of doublings and altered cell morphology compared to mock-treated S. pombe cells (Figures 5b and c). To determine if the reduced cell growth was due to slower S-phase, it was examined whether 8g affects the cell cycle progression of synchronized S. pombe cells (Figure 12). Synchronized cells released from the G2 phase treated with 5 pM 8g showed both delayed (-100 min after release instead of -80 min) and prolonged (120 min instead of 80 min) S-phase compared to mock-treated cells (Figure 5d). Increasing the concentration of 8g to 10 pM resulted in G2-arrested cells that were unable to progress through the cell cycle (Figure 5d).

To more directly examine the effect of 8g on DNA replication, DNA fiber analysis was performed (Figure 5e). The mean DNA replication tract length was significantly shorter in 8g-treated cells compared to mock-treated cells (p = 1.6 ^c KG⁷) (Figure 5e), suggesting that 8g affects DNA replication progression in S. pombe in a STAT3- independent manner.

EXAMPLE 9: SPR screen for STAT3 protein binding

Surface plasmon resonance (SPR) was used to determine binding of compounds to STAT3 in vitro. The SPR screen with the STAT3 protein was performed with 5 mM of each compound. Compared with KC45 (8g), three compounds (KC240 (8h), KC281 (8o), and KC298 (8q)) showed improved binding properties to the STAT3 protein, suggesting that these compounds may be better inhibitors to STAT3 phosphorylation in vivo (Figure 16). KC246 (8j) and KC261 (8n) had similar binding affinity as KC45 (8g) to the STAT3 protein (Figure 16). KC234 (8i), KC235 (8k), KC250 (81), and KC253 (8m) showed less binding than KC45 (8g).

EXAMPLE 10: HeLa Cell viability assay

In a DNA polymerase stop assay (see Example 2, above), several compounds were shown to inhibit DNA synthesis on G4 DNA (Figure 7e) but not DNA synthesis on non-G4 DNA (Figure 7f). These data suggest that these compounds are selective G4 stabilizers. It was determined whether any of the compounds show enhanced toxicity in cancer cells compared to our previous best molecule (KC45 (8g)) by performing cell viability assays of HeLa cells. Cell viability was measured using the PrestoBlue cell viability reagent (Invitrogen). The results of 9 compounds are shown (Figure 17) and tested in triplicates at 6 different concentrations, (0.78; 1.56; 3.12; 6.25; 12.5 and 25 mM). Several of the tested compounds were more effective compared to the reference compounds at the highest concentrations tested, but not at low compound concentrations. Treatment with 25 pM KC246 (8j) and KC298 (8q), show a stronger decrease in HeLa cell viability (5%, 7% respectivly) compared to the reference compound at the same concentration (KC45 (8g) 22%) (Figurel7A and 17C). Whereas compound KC253 (8m) showed compared to KC45 a stronger reduction in cell viability when both 12.5 (15%) and 25 (14%) pM of compound was used (Figure 17B). KC240 (8h) was less toxic to Hela cells at most concentrations tested however at 12.5 pM the compound was comparable in toxicity compared to the reference compound (Figure 17A). KC261 (8n), KC281 (8o) and KC235 (8k) reduced cell viability with about 80% when 1.58 mM was used, whereas KC45 (8g) does not decrease HeLa cell viability at this concentration (Figure 17A and 17B). At higher compound concentrations KC261 (8n), KC281 (8o) and KC235 (8k) are however less or similar effective in reducing cell viability compared to KC45 (8g) (Figure 17A and 17B).

Two compounds (KC250 (81), KC234 (8i)) showed an enhanced toxicity in HeLa cancer cells compared to the reference compound (Figure 17C). The compounds are more effective in reducing cell viability at low (0.78 and 1.56 mM) and high (12.5 and 25 mM) compound concentrations, but not when intermediate compound concentration (3.12 and 6.25 mM) were used (Figure 17C).

EXAMPLE 11 : Cancer cell lines screening using KC45 (8g)

It has been shown that the triple negative breast cancer cell lines are more sensitive to treatment with KC45 (8g) compared to cells derived from healthy breast tissues (Jamroskovic et al., 2020). To test if other cancer types also are sensitive to KC45 treatment, a cancer tissue screen was performed, including 17 different cancer tissues and in total 50 different cancer cell lines (Figure 18). Treatment of cells for 72 hours at different concentrations of KC45 showed that several cancer types are sensitive to KC45 treatment. The top five tissue cancer types were Ovary, Skin, Kidney, Myeloma, and Breast (Figure 18). The IC50 values were determined for the different cancer cell lines treated with KC45. These numbers ranged from 1.6-4.3 mM (Table 4).

EXAMPLE 12: KC45 (8g) impairs replication of CHIKV

The Chikungunya virus (CHIKV) is an alphavirus belonging to the family of the Togaviridae. Like other alphaviruses, it is a mosquito-borne disease. The Chikungunya fever is characterized by a high fever, rashes and a crippling arthralgia, which may last months to years after the infection. To this day, there is no treatment nor vaccines against any of the diseases caused by alphaviruses.

Alphaviruses are positive-strand RNA (+ssRNA) viruses. This means that their genome is composed of mRNA and can readily be translated into proteins upon entry in host cells. The genome is composed of two open reading frames (ORF): the first one being translated into a polyprotein that is then maturated into four non-structural proteins: nsPl, nsP2, nsP3 and nsP4. The main role of these proteins is to replicate the viral RNA genome (Rupp, Sokoloski, Gebhart and Hardy, 2015). The second ORF is expressed at later stages of the infection and encodes a polyprotein, which once maturated, gives the structural proteins forming the viral particle itself.

A particularity of the CHIKV is that it highjacks the plasma membrane of the host cell and forms evaginations called“replication complexes” in which the viral RNA is segregated and replicated (Frolova et ah, 2010). The +ssRNA is replicated through an intermediate negative-strand which is the template used by the viral RNA-dependent RNA polymerase nsP4 to synthesize more of the +ssRNA (Rupp, Sokoloski, Gebhart and Hardy, 2015).

Both positive and negative strands of virus genome have putative G4 sequences (Lavezzo et al, (2018). Therefore, the potential of a G4-stabilizing compound, KC45, and its capacity to impair the replication of the CHIKV, were investigated.

To determine if KC45 is toxic to Baby Hamster Kidney (BHK) cells, the cells were treated with KC45. It was found that after 17h treatment with 5 mM of KC45 more than 50% of the cells survived, suggesting that KC45 was only mildly toxic at low concentrations to BHK cells (Figure 19). After 44h, the cell viability decreased to 30 and 24% for cells treated with 2.5 and 5mM KC45, respectively (Figure 19).

Next, the BHK cells were infected with CHIKV. At 8 hours post-infection, the ability of the virus to replicate was evaluated by determining expression of eGFP in a

fluorescence microscope. As expected, all cells were expressing eGFP in the absence of KC45 (Figure 20). Strikingly, no fluorescence was observed when cells were treated with 2.5 or 5mM of KC45 was added (Figure 20C, Figure 6D). Taking in consideration the moderate toxicity of KC45 in BHK cells, it is concluded that KC45 is indeed able to impair the replication of the CHIKV. REFERENCES

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Table 1

Compound permeability was measured using a Caco-2 cell assay which indicate that both compounds are able to pass the cell membranes. The cut-off P_app (apparent permeability coefficient) values used corresponds to the fraction absorbed in the gut of <20% (P_app value of 0.2 x 10 ⁶ cm/s for low permeable compounds), and >80% (P_app of 1.6 x 10 ⁶ cm/s for high permeable compounds). Thus, 4f has a high permeability and 8g a medium permeability and a high efflux ratio. Therefore, 8g will most likely have limited absorption after oral administration and further optimization of this property may be needed.

Results summary: Cell line: Caco-2, Passage no: P96, Days in culture: 24,

Concentration: 1 mM

Table 2

Oligonucleotides used in the study.

Table 3

Antibodies used in the study.

Table 4

Potency Metrics for KC45 (8g) across 50 cell lines. Potency and efficacy metrics from a screen of 50 cell lines were derived from logistic curves fitted to growth inhibition or inhibition data using Horizon’s Chalice software. Data metrics were determined by chalice and reported where determined.

SEQUENCE LISTING

<110> Sabouri, Nasim

Chorell, Erik

Wanrooij, Sjoerd

<120> COMPOUNDS TARGETING DUAL G-QUADRUPLEX DNA AND STAT3 <130> NP0465

<160> 12

<170> Patentln version 3.5

<210> 1

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 1

tgaaaacatt attaatggcg tcgagcgtcc g

31

<210> 2

<211> 93

<212> DNA

<213> Schizosaccharomyces pombe

<400> 2

gagaccattc aaaaggataa tgtttgtcat ttagtatatg cccctgctcg tcttcccttc 60

tccggacgct cgacgccatt aataatgttt tea

93

<210> 3

<211> 76

<212> DNA

<213> Schizosaccharomyces pombe

<400> 3

tgttttcctt ggggaagggt ggggcatgtt atgggaaggt ggagaeggae gctcgacgcc 60

attaataatg ttttca

76

<210> 4

<211> 81

<212> DNA

<213> Schizosaccharomyces pombe

<400> 4 tacaggttac aggggttaca gggttacagg ggttacaggg ttacaggtta cggacgctcg 60

acgccattaa taatgttttc a

81

<210> 5

<211> 102

<212> DNA

<213> Schizosaccharomyces pombe

<400> 5

gtatcgtaga gggcgtaaac actatggggt tgtcggggta acgcaacgtt ggcaagctgg 60

gaaacaaata ccggacgctc gacgccatta ataatgtttt ca

102

<210> 6

<211> 25

<212> DNA

<213> Schizosaccharomyces pombe

<400> 6

ggggaagggt ggggcatgtt atggg

25

<210> 7

<211> 29

<212> DNA

<213> Schizosaccharomyces pombe

<400> 7

ggggttacag ggttacaggg gttacaggg

29

<210> 8

<211> 24

<212> DNA

<213> Homo sapiens

<400> 8

tgagggtggt gagggtgggg aagg

24

<210> 9

<211> 22

<212> DNA

<213> Homo sapiens

<400> 9

agggagggcg ctgggaggag gg

22 <210> 10

<211> 25

<212> DNA

<213> Schizosaccharomyces pombe

<400> 10

gtggaagtgt ggtgcatgtt atgtg

25

<210> 11

<211> 75

<212> DNA

<213> Homo sapiens

<400> 11

atatatatat tgagggtggt gagggtgggg aaggatatat atatcggacg ctcgacgcca 60

ttaataatgt tttca

75

<210> 12

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> https://doi.org/10.1002/cbic.201100228

<400> 12

ggguuugggu uuggguuugg g

21

Claims

1. A compound of the formula (I) or (II), or a pharmaceutically acceptable salt, tautomer, or ester thereof, for use in the treatment or prophylaxis of a medical condition that can be treated by stabilizing G-quadruplex (G4) DNA structures and inhibiting STAT3 phosphorylation;

wherein R¹ and R², are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted

piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; or R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form

(a) substituted or un substituted phenyl; or

(b) a substituted or unsubstituted cyclic C4-C8 alkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl;

R³ is selected from the group consisting of hydrogen, methoxy, morpholinyl, halogen, amino, diethylamino, piperidinyl, and piperazinyl substituted with 2- aminobenzoyl; R⁴, R⁵, and R⁶ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; and

R⁷ and R⁸ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; or R⁷ and R⁸ together with the ring atoms to which R⁷ and R⁸, respectively, are attached form phenyl; provided that at least one of the conditions (i), (ii) or (iii) is fulfilled:

(i) R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form (a) substituted or unsubstituted phenyl, or (b) a substituted or unsubstituted cyclic C3-C8 alkyl group;

(ii) R⁷ and R⁸ together with the ring atoms to which R⁷ and R⁸, respectively, are attached form phenyl;

(iii) R⁸ is phenyl and at least one of R³-R⁴ is methoxy.

The compound for use according to claim 1 wherein

R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form (a) substituted or unsubstituted phenyl, or (b) a substituted or unsubstituted cyclic C4-C8 alkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl;

The compound for use according to claim 2 wherein R⁵ is methyl.

The compound for use according to claim 2 or 3, said compound being of the formula (I), wherein

R¹ and R² together with the ring atoms to which R¹ and R², respectively, are attached form substituted or unsubstituted phenyl, or a substituted or unsubstituted cyclic alkyl group selected from cyclopentyl and cyclohexyl.

R³, R⁴ and R⁷ are hydrogen;

R⁵ and R⁶ are methyl; R⁸ is selected from the group consisting of methyl and phenyl.

5. The compound for use according to claim 4, said compound being selected from the group consisting of

2-((4,6-di methyl pyri mi din-2-yl)amino)-4-methylbenzo[/?]quinazolin-7-ol

/V-(4,6-dimethylpyrimidin-2-yl)-4-methyl-7,8,9, 10- tetrahydrobenzo[/_?]quinazolin-2-amine

4-methyl-/V-(4-methyl-6-phenylpyrimidin-2-yl)-7,8,9, 10- tetrahydrobenzo[/?]quinazolin-2-amine;

A-(4,6-dimethylpyrimidin-2-yl)-4-methyl-8,9-dihydro-7//- cyclopenta[/?]quinazolin-2-amine

6 The compound for use according to claim 1, said compound having the formula (III):

wherein R¹, R², R⁴, and R⁵ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted

piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; and R³ is selected from the group consisting of hydrogen, methoxy, morpholinyl, halogen, amino, diethylamino, piperidinyl, and piperazinyl substituted with 2- aminobenzoyl.

7. The compound for use according to claim 6 wherein R⁵ is methyl.

8 The compound for use according to claim 6 or 7 wherein

R¹ is hydrogen or methoxy;

R² is hydrogen or morpholinyl;

R⁴ is hydrogen or methoxy; and

R⁵ is methyl.

9. The compound for use according to claim 8, said compound being selected from the group consisting of:

2-((6-methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(li )-one

2-((4-methyl-6-morpholinoquinazolin-2-yl)amino)quinazolin-4(li7)-one

2-((6, 7-dimethoxy-4-m ethyl quinazolin-2-yl)amino)quinazolin-4(l //)-one

2-((4-m ethyl -7-morpholinoquinazolin-2-yl)amino)quinazolin-4(l //)-one

2-((4-methyl-6-(piperidin-l-yl)quinazolin-2-yl)amino)quinazolin-4(li )-one

2-((6-(di ethyl ami no)-4-m ethyl quinazolin-2-yl)amino)quinazolin-4( l //)-one

2-((6-bromo-4-methylquinazolin-2-yl)amino)quinazolin-4( l //)-one

2-((6-chl oro-4-m ethyl quinazolin-2-yl)amino)quinazolin-4(l //)-one

2-((6-fl uoro-4-m ethyl quinazolin-2-yl)amino)quinazolin-4(l //)-one

2-((8-methoxy-4-methylquinazolin-2-yl)amino)quinazolin-4(l//)-one

2-((6-(4-(2-aminobenzoyl)piperazin-l-yl)-4-methylquinazolin-2- yl)amino)quinazolin-4( l//)-one

2-((6-amino-4-methylquinazolin-2-yl)amino)quinazolin-4(l//)-one

10 The compound for use according to claim 1 wherein R⁸ is phenyl and at least one of R^R⁴ is methoxy.

11. The compound for use according to claim 10 wherein R⁵ is methyl.

12. The compound for use according to claim 10 or 11, said compound being of the formula (I), wherein

R¹, R², R³, and R⁴ are independently hydrogen or methoxy, provided that at least one of R¹, R², R³, and R⁴ is methoxy;

R⁵ and R⁶ are methyl; and

R⁷ is hydrogen.

13 The compound for use according to claim 12, said compound being selected from the group consisting of: 7-methoxy-4-m ethyl -A/-(4-m ethyl -6-phenyl pyri mi din-2-yl)quinazolin-2-amine

5, 6, 7-tri methoxy-4-methyl -A^f-(4-methyl -6-phenyl pyri mi din-2-yl)quinazolin-2- amine

6, 7-dimethoxy -4-methyl -N-(4-methyl-6-phenylpyrimidin-2-yl)quinazolin-2- amine

14. The compound according to any one of claims 1 to 13 for use in the treatment or prophylaxis of cancer.

15. The compound for use according to claim 14 wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, brain cancer (malignant glioma tumor cells), pancreas cancer, colon cancer, and lung cancer.

16. The compound according to any one of claims 1 to 13 for use in the treatment or prophylaxis of viral infection. 17. A method for the treatment of cancer, comprising administering to a patient in need of such treatment an effective amount of a compound according any one of claims 1 to 13.

18. A method for the treatment of viral infection, comprising administering to a patient in need of such treatment an effective amount of a compound according any one of claims 1 to 13. 19. A pharmaceutical composition for use against cancer, said pharmaceutical

composition comprising as the active ingredient a therapeutically effective amount of a compound according to any one of claims 1 to 13, in association with at least one pharmaceutically acceptable excipient, carrier or diluent. 20. A pharmaceutical composition for use against viral infection, said pharmaceutical composition comprising as the active ingredient a therapeutically effective amount of a compound according to any one of claims 1 to 13, in association with at least one pharmaceutically acceptable excipient, carrier or diluent.

21 A compound having the formula (III) for use as a fluorescent probe in an in vitro diagnostic method

wherein R¹, R², R³, R⁴, and R⁵ are independently selected from the group consisting of hydrogen, methoxy, morpholinyl, hydroxy, halogen, C1-C3 alkyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted pyrrolidinyl, and optionally substituted phenyl; and provided that at least one of R² and R³ is selected from the group consisting of morpholinyl, amino, diethylamino, optionally substituted piperidinyl, optionally substituted piperazinyl, and optionally substituted pyrrolidinyl.