Structure–activity relationship studies on quinoxalin-2(1H)-one derivatives containing thiazol-2-amine against hepatitis C virus leading to the discovery of BH6870

Abstract

Chronic hepatitis C virus infection represents a serious global public health problem, typically resulting in fibrosis, cirrhosis, and ultimately hepatocellular carcinoma. Based on our previous discovery of lead compound 2 (Liu et al. J Med Chem 54:5747–5768, 2011), 35 new quinoxalinone derivatives were explored in this study. Outline of the structure–activity relationships (SARs) revealed that compound BH6870 (36) showed high anti-HCV potency ($\hbox {EC}_{50} = 0.21\, \upmu \hbox {M}$) and a good cell safety index (SI $= 47.19$). SARs analysis indicated that quinoxalin-2(1H)-one containing a 4-aryl-substituted thiazol-2-amine moiety was optimal for antiviral activity. Introducing a hydrogen-bond acceptor (such as ester or amide group) at the C-3 position of quinoxalin-2(1H)-one was beneficial for the antiviral potency, and especially, N,N-disubstituted amide was far superior to N-monosubstituted amide. Incorporation of more than one halogen (fluorine or chlorine atom) or a strong electron-withdrawing group on the benzene ring of the thiazole-phenyl moiety might reduce electron atmosphere density further and resulted in a dramatical loss of activity. The NH-group of the lactam moiety was clearly required for anti-HCV activity.

Graphical Abstract

Design and synthesis of quinoxalin-2(1H)-one derivatives as new non-nucleoside small-molecule HCV inhibitors. BH6870 (36), showing higher antiviral potency and a good cell safety index, was identified.

Introduction

In 1989, the virus responsible for most transfusion-associated non-A and non-B hepatitis was identified and cloned, and named hepatitis C virus (HCV) [1]. HCV belongs to the Flaviviridae family and is described as having positive-sense, single-stranded RNA genomes [2]. HCV infection typically progresses to a chronic state, may resulting in fibrosis, cirrhosis, and hepatocellular carcinoma, and has emerged as a leading cause for liver transplantation [3]. There are at least six distinct HCV genotypes (genotypes 1–6), of which genotype 1 accounts for the majority of infections in the U.S., Europe, and Asia and has been particularly difficult to treat [4, 5]. Currently, chronic HCV infection represents a serious global public health problem. It has been estimated that 150 million people worldwide are chronically infected with HCV and that approximately 3–4 million people are newly infected each year, with more than 350,000 deaths annually due to hepatitis C-related liver diseases [6].

Fig. 1

Anti-HCV agents in late-stage clinical development or launched

Fig. 2

Key lead series discovered as small-molecule HCV inhibitors

HCV has a small genome encoding six nonstructural proteins, including NS2, NS3, NS4A, NS4B, NS5A, and NS5B. It is believed that the nonstructural (NS) proteins provide the catalytic machinery for viral replication [7]. Until recently, the standard of care (SOC) for HCV infection consists in the combination of nonspecific immune modulatory agent pegylated interferon (PEG-IFN) plus the antiviral agent ribavirin, which have limited efficacy particularly against major genotype 1 [8] and severe side effects. To address these deficiencies, many molecular targets have been pursued in an effort to identify direct-acting antiviral agents (DAAs) as anti-HCV drugs [9–13]. In 2011, the massive efforts devoted to developing new HCV DAAs were rewarded by the approval of the first-generation of HCV NS3/4A protease inhibitors, boceprevir [14–16] and telaprevir (Fig. 1) [17–22]. Consequently, the new SOC for patients infected with genotype 1a or 1b has been put into practice by using a combination of the NS3/4A protease inhibitor boceprevir or telaprevir with PEG-IFN and ribavirin, increasing the rate of sustained virologic response (SVR) to nearly 70 % [23]. Two years later, the second-generation NS3/4A protease inhibitor simeprevir launched in 2013 for use in combination with PEG-IFN and ribavirin for the treatment of genotype 1 chronic HCV patients (Fig. 1) [24–28]. The other small-molecule inhibitors of NS3/4A protease will be available in clinics in the near future (Fig. 1) [29]. For example, faldaprevir [30, 31], vaniprevir [32–34], and asunaprevir [35, 36] are currently in phase III trials. In addition to the NS3/4A protease, the RNA-dependent RNA polymerase NS5B is another attractive target for antiviral therapy. Several classes of inhibitors for NS5B polymerase are in late-stage clinical trials or have been recently approved. The inhibitors of NS5B polymerase include nucleoside (such as sofosbuvir [37, 38] and R7128 [39, 40]) and non-nucleoside polymerase inhibitors (e.g., deleobuvir [41, 42], tegobuvir [43–45]) (Fig. 1). However, the high mutation rate of HCV and the rapid emergence of drug resistance have prompted continuous efforts to discover and develop drugs with high efficacy and novel mechanisms of action [46–48].

Quinoxalin-2(1H)-one derivatives have been first reported by our group [49] to be potent inhibitors against HCV. Considering the potential cytotoxicity of the analogues with a thiourea group such as compound 1, cyclization strategy was carried out to identify 2 as a potent inhibitor of the HCV with lower cell toxicity properties in vitro (Fig. 2). We demonstrated that the thiazole-phenyl moiety was optimal for anti-HCV activity, and steric conformation in the position C-6 was necessary for inhibition of the HCV replicon system. Moreover, there may be a steric limitation at the C-3 position of quinoxalin-2(1H)-one. Consequently, further work aimed at optimization of moieties at the C-3 position and exploration of optimal substituents on the benzene ring of the thiazole-phenyl moiety was undertaken. Herein, we report our findings that led to the discovery of BH6870.

Results and discussion

Chemistry

Preparation of the ester-substituted quinoxalinone analogues is depicted in Scheme 1. The core structure 5 was constructed according to our previous report [49]. In the first step, secondary amines were used as nucleophiles to replace quantitatively one fluorine atom of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) in the presence of N,N-diisopropylethylamine (DIPEA) or potassium carbonate $(\hbox {K}_{2}\hbox {CO}_{3})$. Subsequently, the remaining fluorine was substituted by primary amines to give 4a–f. Then 10 % palladium on activated carbon was utilized to convert the dinitro substrates to diamino intermediates followed by a self-cyclization to obtain key intermediates 5a–f. Accordingly, subsequent nucleophilic addition with benzoyl isothiocyanate in acetone, followed by deprotection of the benzoyl group provided thiourea compounds 7a–f after treatment with potassium carbonate. Finally, for the construction of the thiazole ring, thiourea substrates were utilized to react with suitably substituted 2-bromoacetophenones to afford target compounds 8–19.

Scheme 1

Synthesis of ester-substituted quinoxalinone analogues 8–19. Reagents and conditions: a $\hbox {R}^{1}\hbox {H}$, DIPEA, THF, rt or $\hbox {R}^{1}\hbox {H}$, $\hbox {K}_{2}\hbox {CO}_{3}$, THF, reflux; b $\hbox {NH}_{2}\hbox {CHCO}_{2}\hbox {R}^{2}(\hbox {CH}_{2} \hbox {CH}_{2})_{{n}}\hbox {CO}_{2}\hbox {R}^{2} \cdot$ HCl, DIPEA, THF, reflux; c 10 % Pd/C, $\hbox {NH}_{4}\hbox {COOH}$, THF/EtOH (1:1, v/v), rt; d PhCONCS, acetone, reflux; e $\hbox {K}_{2}\hbox {CO}_{3}$, $\hbox {EtOH/H}_{2}\hbox {O}$ (4:1, v/v), $75\, ^{\circ }\hbox {C}$; f $\hbox {R}^{3}\hbox {COCH}_{2}\hbox {Br}$, EtOH, reflux

Tricyclic quinoxalinone compounds were effectively prepared in a 6-step procedure from DFDNB (Scheme 2). Reaction of monosubstituted intermediate 3a with dimethyl 1H-imidazole-4,5-dicarboxylate in acetone and with potassium carbonate under reflux for 6 h gave smoothly the key 4,6-dinitro intermediate 20. Reductive cyclization of 20 to the corresponding imidazo[1,5-a]quinoxalin-4-one 21 was initially attempted according to the hydrogenation procedures used above. Unfortunately, it was found that the products were a complex mixture of several components, of which only a small amount of the desired compound 21 was observed by HPLC-MS, but the major product was its hydroxylamine intermediate. Finally, it was found that the combination of sodium dithionite [50] in concentrated hydrochloric acid was the effective condition of choice for conducting the reductive cyclization of 20 to 21 and its product of ester hydrolysis, which was next converted conveniently to the desired product 21 after the reaction mixture was filtered and concentrated in vacuo followed by an esterification reaction with methanol in the presence of concentrated sulfuric acid. Finally, the target compounds 24–27 could easily be obtained by the introduction of a thiourea moiety into the free amino group of intermediate 21 and then performing a cyclization in 3 steps following reaction steps d–f in Scheme 1.

Scheme 2

Synthesis of tricyclic quinoxalinone analogues 24–27. Reagents and conditions: a N-methylcyclohexylamine, DIPEA, THF, rt; b dimethyl 1H-imidazole-4,5-dicarboxylate, $\hbox {K}_{2}\hbox {CO}_{3}$, acetone, reflux; c $\hbox {Na}_{2}\hbox {S}_{2}\hbox {O}_{4}$, concd. HCl, $\hbox {MeOH/H}_{2}\hbox {O}$ (1:1, v/v), $70 \, ^{\circ }\hbox {C}$; then concd. $\hbox {H}_{2}\hbox {SO}_{4}$, MeOH, reflux; d PhCONCS, acetone, reflux; e NaOMe, MeOH, $50\, ^{\circ }\hbox {C}$; f $\hbox {R}^{3}\hbox {COCH}_{2}\hbox {Br}$, MeOH, reflux

Scheme 3

Synthesis of amide-substituted quinoxalinone analogues 31–48. Reagents and conditions: a (i) $\hbox {LiOH}\cdot \hbox {H}_{2}\hbox {O}$, $\hbox {EtOH/H}_{2}\hbox {O}$ (5:1, v/v), $50\, ^{\circ }\hbox {C}$; (ii) 2M MeNHR$^{4}$ in THF, EDCI, HOBt, THF, $60\, ^{\circ }\hbox {C}$; b PhCONCS, acetone, reflux; c $\hbox {K}_{2}\hbox {CO}_{3}$, $\hbox {EtOH/H}_{2}\hbox {O}$ (4:1, v/v), $75\, ^{\circ }\hbox {C}$; d $\hbox {R}^{3}\hbox {COCH}_{2}\hbox {Br}$, EtOH, reflux

Amide-substituted quinoxalinones were obtained according to the synthetic route shown in Scheme 3. Treatment of 5 with lithium hydroxide (LiOH) easily gave the corresponding carboxylic acid intermediates. And then, the intermediate was incorporated to commercially available methylamine or dimethylamine solution in tetrahydrofuran using EDCI and HOBt as condensation agents to deliver the amide-substituted intermediates 28a–h. Subsequently, the target compounds 31–48 could be smoothly provided using the aforementioned introduction of the thiourea moiety and cyclization conditions.

Synthesis of 1-N-substituted quinoxalinone 53 was accomplished as outlined in Scheme 4. Treatment of lactam 5d with methyl iodide using potassium carbonate as a base resulted in the 1-N-substituted quinoxalinone intermediate 49 followed by an ester hydrolysis and coupling with dimethylamine to yield amide 50. Accordingly, target compound 53 was readily obtained following the same protocol shown in Scheme 1.

Scheme 4

Synthesis of 1-N-substituted quinoxalinone 53. Reagents and conditions: a $\hbox {CH}_{3}\hbox {I}$, $\hbox {K}_{2}\hbox {CO}_{3}$, acetone, $50\, ^{\circ }\hbox {C}$; b (i) $\hbox {LiOH}\cdot \hbox {H}_{2}\hbox {O}$, $\hbox {EtOH/H}_{2}\hbox {O}$ (5:1, v/v), $50\, ^{\circ }\hbox {C}$; (ii) 2M dimethylamine in THF, EDCI, HOBt, THF, $60\, ^{\circ }\hbox {C}$; c PhCONCS, acetone, reflux; d $\hbox {K}_{2}\hbox {CO}_{3}$, $\hbox {EtOH/H}_{2}\hbox {O}$ (4:1, v/v), $75\, ^{\circ }\hbox {C}$; e $4\hbox {-FC}_{6}\hbox {H}_{4}\hbox {COCH}_{2}\hbox {Br}$, MeOH, reflux

Anti-HCV activity in vitro and SAR studies

The anti-HCV activity and cytotoxicity of synthesized quinoxalinone derivatives were evaluated in the HCV RNA replicon system in Huh7-ET cells, as previously described [49, 51]. The results are currently summarized in Tables 1, 2, 3, 4. rIFN$\upalpha$-2b (recombinant human interferon alfa-2b) is a fused form of IFN$\upalpha$-2b with human serum albumin used as a positive control for inhibition of HCV with an approximate $\hbox {EC}_{50}$ of 0.08 IU/mL [49]. Briefly, the concentration of compound to inhibit HCV RNA replication activity by 50 % $(\hbox {EC}_{50})$, the concentration of compound to decrease cell viability by 50 % $(\hbox {CC}_{50})$, and the selective index (SI) calculated as the $\hbox {CC}_{50}/\hbox {EC}_{50}$ ratio are presented.

Previous studies on structure–activity relationships (SARs) indicated that there may be a steric limitation at the C-3 position of quinoxalin-2(1H)-one [49]. Initially, the hydrogen atom at the C-3 position was replaced by an ester group. These modifications were intended to introduce a hydrogen-bond acceptor and change the distance between the ester group and quinoxalinone skeleton, thus making the resulting compounds show an improved anti-HCV activity. Results of these modifications are summarized in Table 1.

Thus, replacement of hydrogen atom at the C-3 position of 2 with propionate side chain resulted in 8 with $\hbox {EC}_{50} = 7.13\, \upmu \hbox {M}$, fourfold less active than 2 ($\hbox {EC}_{50} = 1.67 \,\upmu \hbox {M}$). Incorporation of a fluorine atom on the phenyl ring of 8 resulted in compound 9 exerting no antiviral activity against HCV. Replacement of N-methylcyclohexylamine at the C-6 position of 8 with N-methylaniline side chain resulted in 10 and 11. Interestingly, fluorine-substituted compound 11 exhibited potent activity against HCV with $\hbox {EC}_{50} = 1.30 \,\upmu \hbox {M}$ similar to that of inhibitor 2. However, introduction of a morpholine group (12 and 13) in the position C-6 was unfavorable for the antiviral potency. This could have been due to the fact that a compound containing hydrophobic groups may effectively improve its binding to the target protein. When the substituent at the C-6 position was kept constant as N-methylcyclohexylamine, decreasing the distance between the ester group and quinoxalinone skeleton resulted in 14 and 15. Similarly, compound 14 bearing a fluorine atom had an excellent cellular potency against HCV with $\hbox {EC}_{50} = 1.14\, \upmu \hbox {M}$. However, enhancing $\uppi -\uppi$ interactions (15) significantly decreased the antiviral activity. Furthermore, neither the introduction of N-methylaniline side chain at the C-6 position (16 and 17) nor morpholine group (18 and 19) provided an obvious potency against HCV or improvement in the selectivity index. Consequently, although no single compound displayed significantly increased potency against the HCV replicon compared to 2, it was encouraging to see this kind of activity with ester-substituted compounds.

Table 1 Inhibitory effects of ester-substituted quinoxalin-2(1H)-one derivatives on HCV replication in Huh7

Previous SARs from our group indicated that the mimic of 3,4-double bond of quinoxalinone by a five-membered ring provided a slight improvement in binding potency [49]. Thus, in order to balance the lipid/water partition coefficient, we selected the hydrophilic imidazole ring to mimic the 3,4-double bond of quinoxalinone and incorporated an ester group on the five-membered ring to further study the effect of steric limitation. Cellular data of compounds with double bond mimics are tabulated in Table 2. Preparation of inhibitor 25, bearing a fluorine atom in the para position, resulted in a slightly lower potency ($\hbox {EC}_{50}= 4.14\,\upmu \hbox {M}$) than that of 14 ($\hbox {EC}_{50 }= 1.14 \,\upmu \hbox {M}$). Attempts to replace the fluorine atom with a chlorine atom (26, $\hbox {EC}_{50 }= 4.94 \upmu \hbox {M}$) or directly remove the halogen (24, $\hbox {EC}_{50 }= 3.94 \,\upmu \hbox {M}$) failed to provide improvement in binding potency. Moreover, enhancing $\uppi -\uppi$ interactions (27, $\hbox {EC}_{50 }= 6.14\, \upmu \hbox {M}$) did not show appreciable binding affinity with the target either. It can be concluded from the data in Table 2 that increase of steric hindrance and hydrophilicity is unfavorable for the anti-HCV activity, but the 3,4-double bond can be to some extent mimicked by a five-membered ring.

Table 2 Inhibitory effects of tricyclic quinoxalinone derivatives on HCV replication in Huh7 cells

Bioisosteres modulate biological activity by virtue of subtle differences in their physicochemical properties. A classical divalent bioisosteric replacement involving two single bonds is –O– with –NH–. This replacement has been widely used in the drug discovery process [52]. On the basis of the observed SARs, it was encouraging to know that replacement of the hydrogen atom at the C-3 position with an ester group preserved antiviral activity against HCV. Consequently, we decided to further explore optimal substituents for the hydrogen atom at the C-3 position by introducing an amide group as ester bioisostere. The effect on the anti-HCV activity of the bioisostere derivatives is shown in Table 3. Initially, retaining N-methylcyclohexylamine as $\hbox {R}^{1}$ group, incorporation of N-substituted propionamide side chain resulted in 31 and 32 with $\hbox {EC}_{50 }= 11.62$ and $3.16 \,\upmu \hbox {M}$, respectively. Introduction of a morpholine group as a replacement of the N-methylcyclohexylamine yielded N-monosubstituted amide 33 and N,N-disubstituted amide 34 with lower binding affinity ($\hbox {EC}_{50 }> 20\, \upmu \hbox {M}$ and $\hbox {EC}_{50 }= 6.03 \,\upmu \hbox {M}$, respectively). Interestingly, it was obviously found that amide types of 32 ($\hbox {EC}_{50 }= 3.16 \,\upmu \hbox {M}$) and 34 ($\hbox {EC}_{50 }= 6.03\, \upmu \hbox {M}$) exhibited significant enhancement in activity compared to the corresponding ester types of 9 ($\hbox {EC}_{50 }> 10 \,\upmu \hbox {M}$) and 12 ($\hbox {EC}_{50 }> 10 \,\upmu \hbox {M}$). Further, the N,N-disubstituted amide inhibitor was far superior to the N-monosubstituted amide compound in antiviral activity against HCV. Similarly, the rules deduced from further results of incorporation of a formamide group were consistent with the data obtained from the introduction of propionamide side chain above. Thus, it was encouraging to see that the antiviral potency of inhibitors 35 and 36 was greatly improved with N-methylcyclohexylamine as $\hbox {R}^{1}$ group with $\hbox {EC}_{50 }= 1.12\, \upmu \hbox {M}$ and $0.21 \,\upmu \hbox {M}$, a fivefold improvement in potency compared to the respective analogues of the corresponding ester type 14. In addition, it was evident that the application of bioisostere provided better selectivity (36, SI = 47.19). When the N-methylaniline moiety was introduced to C-6 position, removal of the fluorine atom from the benzene ring resulted in compounds 37 ($\hbox {EC}_{50 }> 20 \,\upmu \hbox {M}$) and 38 ($\hbox {EC}_{50 }= 1.25 \,\upmu \hbox {M}$), which also demonstrated that N,N-disubstituted amide inhibitor did appreciably improve target binding or cellular activity. However, replacement of phenyl with the naphthalene group yielded compounds 39 and 40 with $\hbox {EC}_{50 }> 20 \,\upmu \hbox {M}$ and $\hbox {EC}_{50 }= 7.78 \,\upmu \hbox {M}$, respectively. This result clearly reinforced the fact that enhancing $\uppi -\uppi$ interactions resulted in loss of potency.

Table 3 Inhibitory effects of amide-substituted quinoxalin-2(1H)-one derivatives on HCV replication in Huh7 cells

In view of the observed SARs above, it was clear that incorporation of the N,N-disubstituted formamide moiety did appreciably improve antiviral activity against HCV. Thus, to further explore amide analogues with high potency and excellent selectivity, compounds bearing a wide variety of halogen substituents attached to the benzene ring were synthesized. N-methylcyclohexylamine was retained as $\hbox {R}^{1}$ side chain because it demonstrated improved activity against HCV in comparison to other R$^{1}$ moieties. Data for the resulting compounds are summarized in Table 4. Moving the fluorine atom to ortho or meta position of phenyl resulted in 42 and 43 with $\hbox {EC}_{50 }= 3.58\, \upmu \hbox {M}$ and $3.08 \,\upmu \hbox {M}$, respectively, a 19- to 22-fold loss in activity compared to inhibitor 36 which exhibited potent activity against HCV ($\hbox {EC}_{50 }= 0.16\, \upmu \hbox {M}$). However, removal of the fluorine atom in 42 gave 41, a slightly more active compound with $\hbox {EC}_{50 }= 1.58 \,\upmu \hbox {M}$. The same effect was observed when incorporating more than one fluorine or chlorine atom on the benzene ring (compounds 44–47). On the other hand, replacement of the hydrogen atom in 41 with a cyano group in the para position of phenyl yielded 48 with similar potency ($\hbox {EC}_{50 }= 2.48\, \upmu \hbox {M}$). All the above data suggested that introduction of more than one halogen (fluorine or chlorine atom) or a strong electron-withdrawing group on the benzene ring might reduce electron atmosphere density further and affect the $\uppi -\uppi$ interactions, and this may contribute to the difference in potency and cytotoxicity. Moreover, based on inhibitor 36, modification by blocking the lactam NH-group of quinoxalin-2(1H)-one was performed to result in 53 with $\hbox {EC}_{50 }= 3.43 \,\upmu \hbox {M}$, with a 21-fold loss in activity, which demonstrated that the NH-group on the lactam moiety was definitely required for anti-HCV activity.

Table 4 Inhibitory effects of N,N-dimethyl formamide substituted quinoxalin-2-one derivatives on HCV replication in Huh7 cells

Conclusions

This study has identified new quinoxalin-2(1H)-one derivatives as potent inhibitors against HCV. On the basis of lead compound 2, 35 new quinoxalinone derivatives were designed and synthesized. The anti-HCV activity and a further SARs study of these compounds were carried out in an effort to lead to the the discovery of BH6870 (36) as a new non-nucleoside small-molecule HCV inhibitor showing high antiviral potency ($\hbox {EC}_{50} = 0.21 \,\upmu \hbox {M}$) and a good cell safety index (SI = 47.19).

In conclusion, quinoxalin-2(1H)-one containing a 4-aryl-substituted thiazol-2-amine moiety was optimal for antiviral activity. Increase of steric hindrance and hydrophilicity was unfavorable for the anti-HCV activity although the 3,4-double bond could be to some extent mimicked by a five-membered ring. Introducing a hydrogen-bond acceptor (such as ester or amide group) at the C-3 position of quinoxalin-2(1H)-one was beneficial for the antiviral potency, especially when N,N-disubstituted amide was far superior to N-monosubstituted amide. Moreover, incorporation of more than one halogen (fluorine or chlorine atom) or a strong electron-withdrawing group on the benzene ring of thiazole-phenyl moiety might reduce electron atmosphere density further and resulted in a dramatical loss of potency, and the NH-group of the lactam moiety was clearly required for anti-HCV activity.

Experimental

Chemistry

General All of the chemicals used were obtained from commercial sources (Acros Organics, Alfa Aesar, and Sigma-Aldrich), and were used without further purification. Solvents were used without purification or drying, unless otherwise noted. $^{1}\hbox {H}$ NMR and $^{13}\hbox {C}$ NMR spectra were recorded at 300 and 150 MHz, respectively, on Varian Mercury and Varian NMR System spectrometers in acetone-d6 or DMSO-d6 with TMS as the internal standard. Chemical shifts were reported in ppm ($\updelta$) relative to the solvent, and coupling constants (J) were reported in Hz. Melting points were determined without correction with a Yanaco micromelting point apparatus. Automatic HPLC-MS analysis was performed on a Thermo Finnigan LCQ Advantage mass spectrometer equipped with an Agilent pump, an Agilent detector, an Agilent liquid handler, and a fluent splitter. The column used was a Kromasil C18 column ($4.6 \,\upmu \hbox {m}$, $4.6\hbox { mm} \times 50\hbox { mm}$) from DIKMA for analysis. The eluent was a mixture of acetonitrile and water containing 0.05 % HCOOH with a linear gradient from 5:95 to 95:5 (v/v) of acetonitrile–$\hbox {H}_{2}\hbox {O}$ within 5 min at a 1 mL/min flow rate for analysis. The UV detection was carried out at a UV wavelength of 254 nm. The 5 % of the eluent was split into the MS system. Mass spectra were recorded in either positive or negative ion mode using electrospray ionization (ESI). High-resolution mass spectra (HRMS) were recorded on Thermo Scientific Exactive Plus System. The ion source is ESI. Flash column chromatography was performed with silica gel 60 (200–300 mesh) from Qingdao Haiyang Chemical Co., Ltd. All tested compounds were purified until the purity was $\ge$95 %, detected by HPLC-MS under UV 254 nm wavelength, NMR, melting point, and HRMS.

General procedure A for the synthesis of compounds 4a, 4c, 4d, and 4f

To a stirred solution of 1,5-difluoro-2,4-dinitrobenzene (DFDNB; 2.04 g, 10 mmol) in THF (50 mL) was added DIPEA (1.75 mL, 10 mmol) and N-methylcyclohexylamine (1.30 mL, 10 mmol) or morpholine (0.87 mL, 10 mmol). After vigorously stirring at room temperature until the total disappearance of DFDNB monitored by HPLC-MS analysis, intermediate (3a or 3c) was obtained without purification. Then, DIPEA (3.50 mL, 20 mmol) and L-glutamic acid dimethyl ester hydrochloride (2.12 g, 10 mmol) or diethyl aminomalonate hydrochloride (2.12 g, 10 mmol) were added and stirred under reflux for 6–36 h. After the reaction was complete (monitored by HPLC-MS analysis), the solvent was evaporated in vacuo. Saturated ammonium chloride solution (50 mL) was added to the resulting product and was further extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 50 mL). The organic layers were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo. Intermediate compound (4a, 4c, 4d or 4f) was obtained after purification by silica gel column chromatography.

Dimethyl 2-((5-(cyclohexyl(methyl)amino)-2,4-dinitrophenyl)amino)pentanedioate (4a)

Following general procedure A starting from N-methylcyclohexylamine (1.30 mL, 10 mmol) yielded an orange oil (3.75 g, 83 % over two steps); $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta = 8.70-8.67$ (m, 2H), 6.23 (s, 1H), 4.65 (q, J = 6.9 Hz, 1H), 3.80 (s, 3H), 3.62 (s, 3H), 3.49–3.41 (m, 1H), 2.80 (s, 3H), 2.54 (t, J = 7.2 Hz, 2H), 2.29 (q, J = 6.9 Hz, 2H), 1.86–1.82 (m, 4H), 1.72–1.59 (m, 3H), 1.49–1.08 ppm (m, 3H); HRMS-ESI $m/z\,[\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{29}\hbox {N}_{4}\hbox {O}_{8}$: 453.1980, found: 453.1976.

Dimethyl 2-((5-morpholino-2,4-dinitrophenyl)amino)pentanedioate (4c)

Following general procedure A starting from morpholine (0.87 mL, 10 mmol) yielded a dark-yellow powder (3.28 g, 77 % over two steps); mp: 142–144 $^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta$ = 8.87 (d, J = 7.2 Hz, 1H), 8.83 (s, 1H), 6.46 (s, 1H), 4.78 (q, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.78 (t, J = 4.5 Hz, 4H), 3.62 (s, 3H), 3.24 (t, J = 4.5 Hz, 4H), 2.53 (t, J = 7.2 Hz, 2H), 2.28 ppm (q, J = 7.2 Hz, 2H); HRMS-ESI $m/z\,[M + \hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{23}\hbox {N}_{4}\hbox {O}_{9}$: 427.1460, found: 427.1456.

Diethyl 2-((5-(cyclohexyl(methyl)amino)-2,4-dinitrophenyl)amino)malonate (4d)

Following general procedure A starting from N-methylcyclohexylamine (1.30 mL, 10 mmol) yielded a dark-yellow powder (3.62 g, 80 % over two steps); mp: 123–124 $^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta$ = 9.13 (d, J = 6.0 Hz, 1H), 8.71 (s, 1H), 6.19 (s, 1H), 5.31 (d, J = 6.0 Hz, 1H), 4.42–4.25 (m, 4H), 3.53–3.43 (m, 1H), 2.78 (s, 3H), 1.88–1.84 (m, 4H), 1.72–1.60 (m, 3H), 1.52–1.36 (m, 2H), 1.29 (t, J = 7.2 Hz, 6H), 1.24–1.06 ppm (m, 1H); HRMS-ESI $m/z\,[\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{29}\hbox {N}_{4}\hbox {O}_{8}$: 453.1980, found: 453.1978.

Diethyl 2-((5-morpholino-2,4-dinitrophenyl)amino)malonate (4f)

Following general procedure A starting from morpholine (0.87 mL, 10 mmol) yielded a dark-yellow powder (3.07 g, 72 % over two steps); mp: 121–122 $^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta$ = 9.20–9.18 (m, 1H), 8.84 (s, 1H), 6.34 (s, 1H), 5.41–5.39 (m, 1H), 4.38–4.28 (m, 4H), 3.79 (t, J = 4.5 Hz, 4H), 3.23 (t, J = 4.5 Hz, 4H), 1.29 ppm (t, J = 7.2 Hz, 6H); HRMS-ESI $m/z\,[\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{23}\hbox {N}_{4}\hbox {O}_{9}$: 427.1460, found: 427.1458.

General procedure B for the synthesis of compounds 4b and 4e

To a stirred solution of 1,5-difluoro-2,4-dinitrobenzene (DFDNB; 2.04 g, 10 mmol) in THF (50 mL) was added $\hbox {K}_{2}\hbox {CO}_{3}$ (1.38 g, 10 mmol) and N-methylaniline (1.08 mL, 10 mmol). The mixture was stirred under reflux for 36 h. The suspension was cooled and filtered. Then, the filtrate was concentrated under reduced pressure. The residue was dissolved in saturated ammonium chloride solution (50 mL) and extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 50 mL). The organic phases were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo to give crude intermediate 3b. Then, the residue was dissolved in THF (50 mL). DIPEA (3.50 mL, 20 mmol) and L-glutamic acid dimethyl ester hydrochloride (2.12 g, 10 mmol) or diethyl aminomalonate hydrochloride (2.12 g, 10 mmol) were added and stirred under reflux for 12–24 h. After the reaction was completed (monitored by HPLC-MS analysis), the solvent was evaporated in vacuo. Saturated ammonium chloride solution (50 mL) was added to the resulting product and was further extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 50 mL). The organic layers were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo. Intermediate compound (4b or 4e) was obtained after purification by silica gel column chromatography.

Dimethyl 2-((5-(methyl(phenyl)amino)-2,4-dinitrophenyl)amino)pentanedioate (4b)

Following general procedure B yielded an orange oil (3.35 g, 75 % over two steps); $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta$ = 8.90 (d, J = 6.9 Hz, 1H), 8.70 (s, 1H), 7.32–7.26 (m, 2H), 7.09–7.04 (m, 3H), 6.76 (s, 1H), 4.82–4.76 (m, 1H), 3.82 (s, 3H), 3.62 (s, 3H), 3.52 (s, 3H), 2.54 (t, J = 6.9 Hz, 2H), 2.32 ppm (q, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{23}\hbox {N}_{4}\hbox {O}_{8}$: 447.1510, found: 447.1508.

Diethyl 2-((5-(methyl(phenyl)amino)-2,4-dinitrophenyl)amino)malonate (4e)

Following general procedure B yielded an orange powder (3.03 g, 68 % over two steps); mp: 118–119 $^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, Acetone-d6): $\updelta$ = 9.24 (d, J = 6.3 Hz, 1H), 8.69 (s, 1H), 7.32–7.27 (m, 2H), 7.12–7.07 (m, 3H), 6.60 (s, 1H), 5.39 (d, J = 6.3 Hz, 1H), 4.41–4.25 (m, 4H), 3.49 (s, 3H), 1.29 ppm (t, J = 7.2 Hz, 6H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{23}\hbox {N}_{4}\hbox {O}_{8}$: 447.1510, found: 447.1507.

General procedure C for the synthesis of compounds 5a–f

Compound 4 (3 g) was dissolved in a mixed solvent of THF (50 mL) and EtOH (50 mL), followed by the addition of 10 % Pd/C (3 g) and $\hbox {NH}_{4}\hbox {COOH}$ (6 g). The mixture was stirred at room temperature for 2 h. After the reaction was complete (monitored by HPLC-MS analysis), the residue solid was filtered off, and the filtrate was concentrated in vacuo. Key intermediate 5 was obtained after purification by silica gel column chromatography.

Methyl 3-(6-amino-7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl) propanoate (5a)

Following general procedure C starting from 4a (3 g, 6.64 mmol) yielded a brown-yellow powder (2.07 g, 87 %); $\hbox {mp:}~ 207{-}208\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.94 (s, 1H), 7.16 (s, 1H), 6.46 (s, 1H), 5.55 (s, 2H), 3.60 (s, 3H), 2.93 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 7.2 Hz, 2H), 2.69–2.66 (m, 1H), 2.56 (s, 3H), 1.77–1.69 (m, 4H), 1.56–1.52 (m, 1H), 1.38–1.04 ppm (m, 5H); HRMS-ESI $m/z\, [M+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{27}\hbox {N}_{4}\hbox {O}_{3}$: 359.2078, found: 359.2074.

Methyl 3-(6-amino-7-(methyl(phenyl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl) propanoate (5b)

Following general procedure C starting from 4b (3 g, 6.73 mmol) yielded a light-yellow powder (1.99 g, 84 %); $\hbox {mp:}~ 183{-}185\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta = 12.04$ (s, 1H), 7.16–7.11 (m, 3H), 6.68 (t, J = 7.5 Hz, 1H), 6.58–6.56 (m, 3H), 5.67 (s, 2H), 3.56 (s, 3H), 3.11 (s, 3H), 2.92 (t, J = 6.9 Hz, 2H), 2.69 ppm (t, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{21}\hbox {N}_{4}\hbox {O}_{3}$: 353.1608, found: 353.1606.

Methyl 3-(6-amino-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)propaneate (5c)

Following general procedure C starting from 4c (3 g, 7.04 mmol) yielded a brown-yellow powder (1.80 g, 77 %); mp: $>$300$\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.97 (s, 1H), 7.11 (s, 1H), 6.48 (s, 1H), 5.64 (s, 2H), 3.76 (t, J = 4.2 Hz, 4H), 3.60 (s, 3H), 2.93 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 4.2 Hz, 4H), 2.72 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{16}\hbox {H}_{21}\hbox {N}_{4}\hbox {O}_{4}$: 333.1557, found: 333.1558.

Ethyl 6-amino-7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (5d)

Following general procedure C starting from 4d (3 g, 6.64 mmol) yielded an orange powder (1.96 g, 86 %); $\hbox {mp:} 214-216\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.30 (s, 1H), 7.28 (s, 1H), 6.46 (s, 1H), 6.15 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 2.74–2.68 (m, 1H), 2.57 (s, 3H), 1.79–1.69 (m, 4H), 1.56–1.52 (m, 1H), 1.38–1.05 ppm (m, 8H); HRMS-ESI $m/z\, [M + \hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{25}\hbox {N}_{4}\hbox {O}_{3}$: 345.1921, found: 345.1917.

Ethyl 6-amino-7-(methyl(phenyl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (5e)

Following general procedure C starting from 4e (3 g, 6.73 mmol) yielded an orange powder (1.82 g, 80 %); $\hbox {mp:}~ 132{-}134\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.40 (s, 1H), 7.32 (s, 1H), 7.15 (t, J = 7.8 Hz, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.60 (d, J = 8.1 Hz, 2H), 6.56 (s, 1H), 6.26 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 3.12 (s, 3H), 1.27 ppm (t, J = 7.2 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{19}\hbox {N}_{4}\hbox {O}_{3}$: 339.1452, found: 339.1446.

General procedure D for the synthesis of compounds 7a–f, 30a–h, and 52

To a stirred solution of amine intermediate (5, 28 or 50, 2.0 mmol) in dry acetone (30 mL), benzoyl isothiocyanate (0.32 mL, 2.4 mmol) was added. The reaction mixture was refluxed for 3 h. After the reaction was completed, the solvent was evaporated in vacuo to give crude compound (6, 29 or 51) without further purification. Then, the residue and $\hbox {K}_{2}\hbox {CO}_{3}$ (415 mg, 3.0 mmol) were dissolved in a mixed solvent of EtOH (40 mL) and $\hbox {H}_{2}\hbox {O}$ (10 mL). The reaction mixture was heated at $75\, ^{\circ }\hbox {C}$ for 1 h. After the reaction was complete (monitored by HPLC-MS analysis), the solvent was evaporated carefully under reduced pressure. The thiourea intermediate (7, 30 or 52) was characterized after purification by silica gel column chromatography.

Methyl 3-(7-(cyclohexyl(methyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl)propanoate (7a)

Following general procedure D starting from 5a (716 mg, 2.0 mmol) yielded a dark-yellow powder (584 mg, 70 % over two steps); $\hbox {mp:}~ 117{-}119\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.23 (s, 1H), 9.12 (s, 1H), 8.17 (s, 1H), 7.93 (s, 2H), 7.37 (s, 1H), 3.60 (s, 3H), 3.02 (t, J = 7.2 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.72–2.68 (m, 1H), 2.63 (s, 3H), 1.80–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.01 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{28}\hbox {N}_{5}\hbox {O}_{3}$S: 418.1907, found: 418.1902.

Methyl 3-(7-(methyl(phenyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl) propanoate (7b)

Following general procedure D starting from 5b (704 mg, 2.0 mmol) yielded a yellow powder (534 mg, 65 % over two steps); $\hbox {mp:}~ 125{-}127\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.37 (s, 1H), 9.21 (s, 1H), 8.43 (s, 1H), 7.86 (br. s, 2H), 7.36 (s, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 6.67 (d, J = 8.1 Hz, 2H), 3.56 (s, 3H), 3.11 (s, 3H), 3.01 (t, J = 6.9 Hz, 2H), 2.73 ppm (t, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{22}\hbox {N}_{5}\hbox {O}_{3}$S: 412.1438, found: 412.1437.

General procedure E for the synthesis of compounds 8–19, 24–27, 31–48, and 53

Thiourea intermediate (7, 23, 30 or 52, 0.20 mmol) and various $\alpha$-bromo-substituted ketones (0.24 mmol) were dissolved in EtOH (6 mL) and refluxed until the total disappearance of thiourea intermediate monitored by HPLC-MS analysis. The mixture was concentrated in vacuo, and the final products 8–19, 24–27, 31–48, and 53 were characterized after purification by silica gel column chromatography.

Methyl 3-(7-(cyclohexyl(methyl)amino)-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxalin-2-yl)propanoate (8)

Following general procedure E starting from 7a (83 mg, 0.20 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded a yellow powder (74 mg, 72 %); $\hbox {mp:}~ 234{-}236\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.68 (s, 1H), 9.69 (s, 1H), 8.58 (s, 1H), 8.10 (d, J = 7.5 Hz, 2H), 7.49–7.44 (m, 4H), 7.34 (t, J = 7.5 Hz, 1H), 3.62 (s, 3H), 3.03 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.73 (br. s, 1H), 2.66 (s, 3H), 1.89–1.85 (m, 2H), 1.71–1.67 (m, 2H), 1.54–1.51 (m, 1H), 1.31–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{32}\hbox {N}_{5}\hbox {O}_{3}$S: 518.2220, found: 518.2218.

Methyl 3-(7-(cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (9)

Following general procedure E starting from 7a (83 mg, 0.20 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a yellow powder (80 mg, 75 %); $\hbox {mp:}~ 247{-}249\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.66 (s, 1H), 9.71 (s, 1H), 8.56 (s, 1H), 8.13 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.47 (s, 1H), 7.43 (s, 1H), 7.26 (t, J = 9.0 Hz, 2H), 3.61 (s, 3H), 3.01 (t, J = 7.2 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.71 (br. s, 1H), 2.65 (s, 3H), 1.87–1.83 (m, 2H), 1.69–1.66 (m, 2H), 1.53–1.50 (m, 1H), 1.29–1.00 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{31}\hbox {FN}_{5}\hbox {O}_{3}\hbox {S}$: 536.2126, found: 536.2122.

Methyl 3-(7-(methyl(phenyl)amino)-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihy droquinoxalin-2-yl)propanoate (10)

Following general procedure E starting from 7b (82 mg, 0.20 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded a brown-yellow powder (85 mg, 83 %); $\hbox {mp:}~ 234{-}236\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.80 (s, 1H), 10.06 (s, 1H), 8.87 (s, 1H), 8.11 (d, J = 7.2 Hz, 2H), 7.49–7.43 (m, 3H), 7.35–7.31 (m, 2H), 7.16 (t, J = 7.8 Hz, 2H), 6.73 (t, J = 7.2 Hz, 1H), 6.62 (d, J = 7.8 Hz, 2H), 3.56 (s, 3H), 3.14 (s, 3H), 3.00 (t, J = 6.9 Hz, 2H), 2.73 ppm (t, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{26}\hbox {N}_{5}\hbox {O}_{3}\hbox {S}$: 512.1751, found: 512.1746.

Methyl 3-(6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-7-(methyl(phenyl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (11)

Following general procedure E starting from 7b (82 mg, 0.20 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a brown-yellow powder (84 mg, 79 %); $\hbox {mp:}~ 253{-}254\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.80 (s, 1H), 10.08 (s, 1H), 8.86 (s, 1H), 8.16 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.42 (s, 1H), 7.35 (s, 1H), 7.27 (t, J = 9.0 Hz, 2H), 7.16 (t, J = 7.8 Hz, 2H), 6.73 (t, J = 7.2 Hz, 1H), 6.62 (d, J = 8.1 Hz, 2H), 3.56 (s, 3H), 3.13 (s, 3H), 3.01 (t, J = 6.9 Hz, 2H), 2.73 ppm (t, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{25}\hbox {FN}_{5}\hbox {O}_{3}\hbox {S}$: 530.1657, found: 530.1653.

Methyl 3-(6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (12)

Following general procedure E starting from 7c (78 mg, 0.20 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a yellow powder (90 mg, 88 %); $\hbox {mp:}~ 254{-}256\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.65 (s, 1H), 9.59 (s, 1H), 8.57 (s, 1H), 8.12 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.45 (s, 1H), 7.42 (s, 1H), 7.26 (t, J = 9.0 Hz, 2H), 3.86 (br. s, 4H), 3.61 (s, 3H), 3.02 (t, J = 6.9 Hz, 2H), 2.85 (br. s, 4H), 2.77 ppm (t, J = 6.9 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{25}\hbox {H}_{25}\hbox {FN}_{5}\hbox {O}_{4}\hbox {S}$: 510.1606, found: 510.1604.

Methyl 3-(7-morpholino-6-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (13)

Following general procedure E starting from 7c (78 mg, 0.20 mmol) and 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded a brown-yellow powder (76 mg, 70 %); $\hbox {mp:}~ 236{-}237\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.35 (s, 1H), 9.57 (s, 1H), 8.42–8.38 (m, 1H), 8.26 (s, 1H), 8.00–7.94 (m, 2H), 7.88 (d, J = 6.3 Hz, 1H), 7.62–7.53 (m, 3H), 7.41 (s, 1H), 7.26 (s, 1H), 3.87 (br. s, 4H), 3.60 (s, 3H), 2.98 (t, J = 7.2 Hz, 2H), 2.88 (br. s, 4H), 2.75 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{29}\hbox {H}_{28}\hbox {N}_{5}\hbox {O}_{4}\hbox {S}$: 542.1856, found: 542.1853.

Ethyl 7-(cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (14)

Following general procedure E starting from 7d (81 mg, 0.20 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded an orange powder (84 mg, 81 %); $\hbox {mp:}~ 184{-}186\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.14 (s, 1H), 9.96 (s, 1H), 8.66 (s, 1H), 8.16 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.65 (s, 1H), 7.52 (s, 1H), 7.29 (t, J = 9.0 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 2.78–2.70 (m, 1H), 2.66 (s, 3H), 1.90–1.87 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.24–1.06 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 163.85, 161.92, 161.72 (d, $^{1}J_{C-F}$ = 243.2 Hz), 153.07, 149.10, 145.24, 141.37, 137.73, 131.88, 130.81 (d, $^{4}J_{C-F}$ = 2.4 Hz), 128.10 (d, $^{3}J_{C-F}$ = 7.6 Hz), 125.69, 123.92, 115.37 (d, $^{2}J_{C-F}$ = 21.2 Hz), 105.26, 100.76, 62.02, 61.28, 38.46, 29.11, 25.50, 24.99, 14.05 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{29}\hbox {FN}_{5}\hbox {O}_{3}\hbox {S}$: 522.1970, found: 522.1971.

Ethyl 7-(cyclohexyl(methyl)amino)-6-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (15)

Following general procedure E starting from 7d (81 mg, 0.20 mmol) and 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded an orange powder (83 mg, 75 %); $\hbox {mp:}~ 132{-}134\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.85 (s, 1H), 9.92 (s, 1H), 8.40–8.38 (m, 2H), 8.03–7.90 (m, 3H), 7.65–7.55 (m, 4H), 7.33 (s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 2.82–2.75 (m, 1H), 2.69 (s, 3H), 1.93–1.89 (m, 2H), 1.74–1.70 (m, 2H), 1.57–1.54 (m, 1H), 1.33–1.05 ppm (m, 8H); $^{13}\hbox {C}$ NMR (150 MHz, DMSO-d6): $\updelta$ = 163.82, 161.59, 152.82, 149.45, 145.29, 141.40, 137.80, 133.49, 132.68, 131.87, 130.64, 128.31, 128.23, 127.46, 126.48, 125.87, 125.68, 125.51, 125.48, 123.87, 109.64, 100.59, 61.91, 61.24, 38.27, 29.14, 25.50, 25.01, 14.03 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{31}\hbox {H}_{32}\hbox {N}_{5}\hbox {O}_{3}\hbox {S}$: 554.2220, found: 554.2219.

Ethyl 7-(methyl(phenyl)amino)-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxylate (16)

Following general procedure E starting from 7e (79 mg, 0.20 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded a dark-yellow powder (62 mg, 62 %); $\hbox {mp:} 255{-}257\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.26 (s, 1H), 10.26 (s, 1H), 8.92 (s, 1H), 8.12 (d, J = 7.2 Hz, 2H), 7.54–7.45 (m, 4H), 7.34 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.8 Hz, 2H), 6.75 (t, J = 7.2 Hz, 1H), 6.65 (d, J = 7.8 Hz, 2H), 4.31 (q, J = 7.2 Hz, 2H), 3.15 (s, 3H), 1.29 ppm (t, J = 7.2 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{3}\hbox {S}$: 498.1594, found: 498.1590.

Ethyl 7-(methyl(phenyl)amino)-6-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17)

Following general procedure E starting from 7e (79 mg, 0.20 mmol) and 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded an orange powder (83 mg, 76 %); $\hbox {mp:}~ 135{-}137\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.95 (s, 1H), 10.27 (s, 1H), 8.73 (s, 1H), 8.39–8.36 (m, 1H), 8.02–7.92 (m, 3H), 7.63 (t, J = 7.5 Hz, 1H), 7.57–7.53 (m, 3H), 7.28 (s, 1H), 7.19 (t, J = 7.8 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 3.18 (s, 3H), 1.27 ppm (t, J = 7.2 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{31}\hbox {H}_{26}\hbox {N}_{5}\hbox {O}_{3}\hbox {S}$: 548.1751, found: 548.1749.

Ethyl 7-morpholino-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxylate (18)

Following general procedure E starting from 7f (75 mg, 0.20 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded an umber powder (67 mg, 70 %); $\hbox {mp:}~ 263{-}265\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.13 (s, 1H), 9.80 (s, 1H), 8.67 (s, 1H), 8.10 (d, J = 7.5 Hz, 2H), 7.60 (s, 1H), 7.55 (s, 1H), 7.47 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 3.89 (br. s, 4H), 2.88 (br. s, 4H), 1.32 ppm (t, J = 7.2 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{24}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{4}\hbox {S}$: 478.1544, found: 478.1542.

Ethyl 6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-7-morpholino-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (19)

Following general procedure E starting from 7f (75 mg, 0.20 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded an orange powder (77 mg, 78 %); mp: 264–266$\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.13 (s, 1H), 9.82 (s, 1H), 8.67 (s, 1H), 8.15 (dd, J = 8.4 Hz, 5.7 Hz, 2H), 7.60 (s, 1H), 7.53 (s, 1H), 7.28 (t, J = 8.7 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 3.89 (br. s, 4H), 2.88 (br. s, 4H), 1.32 ppm (t, J = 7.2 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{24}\hbox {H}_{23}\hbox {FN}_{5}\hbox {O}_{4}\hbox {S}$: 496.1449, found: 496.1445.

Dimethyl 1-(5-(cyclohexyl(methyl)amino)-2,4-dinitrophenyl)-1H-imidazole-4,5-dicarboxylate (20)

To a stirred solution of 1,5-difluoro-2,4-dinitrobenzene (DFDNB; 2.04 g, 10 mmol) in THF (50 mL) was added DIPEA (1.75 mL, 10 mmol) and N-methylcyclohexylamine (1.30 mL, 10 mmol). The reaction mixture was stirred at room temperature until the total disappearance of DFDNB monitored by HPLC-MS analysis. The solvent was evaporated under reduced pressure, and the oily residue was dissolved in saturated ammonium chloride solution (50 mL) and extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 50 mL). The organic phases were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo to give crude intermediate 3a. Then, the residue was dissolved in acetone (70 mL). Dimethyl 1H-imidazole-4,5-dicarboxylate (1.84 g, 10 mmol) and $\hbox {K}_{2}\hbox {CO}_{3}$ (2.76 g, 20 mmol) were added and stirred under reflux for 6 h. After the reaction was complete (monitored by HPLC-MS analysis), the solvent was evaporated in vacuo. Saturated ammonium chloride solution (50 mL) was added to the resulting product and was further extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 50 mL). The organic layers were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo. Intermediate compound 20 was obtained after purification by silica gel column chromatography as a dark-yellow powder (3.32 g, 72 % over two steps); $\hbox {mp:}~ 165-167\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 8.72 (s, 1H), 8.22 (s, 1H), 7.60 (s, 1H), 3.86 (s, 3H), 3.82–3.75 (m, 1H), 3.68 (s, 3H), 2.74 (s, 3H), 1.78–1.56 (m, 7H), 1.44–1.06 ppm (m, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{8}$: 462.1619, found: 462.1618.

Methyl 7-amino-8-(cyclohexyl(methyl)amino)-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-3-carboxylate (21)

To a stirred solution of intermediate 20 (4.61 g, 10 mmol) in a mixed solvent of MeOH (150 mL) and $\hbox {H}_{2}\hbox {O}$ (150 mL) was added sodium dithionite (17.41 g, 100 mmol), and the mixture was heated at $70\, ^{\circ }\hbox {C}$. Concentrated hydrochloric acid (10 mL) was added drop-wise, and the reaction mixture was stirred at $70\, ^{\circ }\hbox {C}$ for 1 h. After the reaction was complete (monitored by HPLC-MS analysis), the reaction mixture was cooled and filtered. The filtrate was concentrated under reduced pressure to give a yellow solid residue, which was then dissolved in MeOH (300 mL), acidified to pH 1–2 with concentrated sulfuric acid, and stirred under reflux for 4 h. The reaction mixture was cooled, neutralized using 2 M sodium hydroxide solution with good stirring, and concentrated in vacuo to give a brown-green solid residue, which was purified by silica gel column chromatography to give the key intermediate 21 as a brown-green powder (3.21 g, 87 %); $\hbox {mp:}~ 258{-}260\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.32 (s, 1H), 9.02 (s, 1H), 7.82 (s, 1H), 6.59 (s, 1H), 5.26 (br. s, 2H), 3.82 (s, 3H), 2.80–2.73 (m, 1H), 2.62 (s, 3H), 1.80–1.70 (m, 4H), 1.56–1.51 (m, 1H), 1.40–1.06 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{3}$: 370.1874, found: 370.1875.

General procedure F for the synthesis of compounds 24–27

To a stirred solution of 21 (1.85 g, 5 mmol) in 80 mL of dry acetone, benzoyl isothiocyanate (0.81 mL, 6 mmol) was added. The reaction mixture was refluxed for 3 h. After the reaction was completed, the solvent was evaporated in vacuo to give crude compound 22 without further purification. Then, the residue was dissolved in MeOH (30 mL), and the mixture was heated at $50\, ^{\circ }\hbox {C}$. Sodium methoxide (0.54 g, 10 mmol) in methanol (50 mL) was added drop-wise, and the reaction mixture was stirred at $50\, ^{\circ }\hbox {C}$ for 1 h. The reaction mixture was cooled, neutralized using 5 % hydrochloric acid, and concentrated under reduced pressure to give a yellow solid residue, which was purified by silica gel column chromatography. Intermediate compound 23 was obtained as a yellow powder in 76 % yield over two steps. Finally, thiourea compound 23 (86 mg, 0.20 mmol) and various $\alpha$-bromo-substituted ketones (0.24 mmol) were dissolved in MeOH (6 mL) and refluxed until the total disappearance of 23 monitored by HPLC-MS analysis. The reaction mixture was concentrated in vacuo, and the final products 24–27 were characterized after purification by silica gel column chromatography.

Methyl 8-(cyclohexyl(methyl)amino)-4-oxo-7-((4-phenylthiazol-2-yl)amino)-4,5-dihydroimidazo[1,5-a]quinoxaline-3-carboxylate (24)

Following general procedure F starting from 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded a yellow powder (86 mg, 81 %); $\hbox {mp:}~ 248{-}250\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.96 (s, 1H), 9.53 (s, 1H), 9.16 (s, 1H), 8.56 (s, 1H), 8.15 (s, 1H), 8.10 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.43 (s, 1H), 7.34 (t, J = 7.2 Hz, 1H), 3.85 (s, 3H), 2.82–2.75 (m, 1H), 2.71 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.69 (m, 2H), 1.55–1.52 (m, 1H), 1.36–1.04 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 162.62, 162.46, 153.17, 150.15, 136.98, 136.37, 134.33, 133.31, 131.96, 128.54, 127.53, 126.29, 126.05, 121.64, 113.54, 111.79, 104.25, 103.99, 62.03, 51.82, 38.15, 29.27, 25.48, 25.02 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{29}\hbox {N}_{6}\hbox {O}_{3}\hbox {S}$: 529.2016, found: 529.2014.

Methyl 8-(cyclohexyl(methyl)amino)-7-((4-(4-fluorophenyl)thiazol-2-yl)amino)-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-3-carboxylate (25)

Following general procedure F starting from 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a light-yellow powder (85 mg, 78 %); $\hbox {mp:}~ 262{-}264\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.95 (s, 1H), 9.55 (s, 1H), 9.16 (s, 1H), 8.57 (s, 1H), 8.17–8.12 (m, 3H), 7.41 (s, 1H), 7.28 (t, J = 9.0 Hz, 2H), 3.85 (s, 3H), 2.81–2.74 (m, 1H), 2.70 (s, 3H), 1.91–1.87 (m, 2H), 1.73–1.69 (m, 2H), 1.55–1.52 (m, 1H), 1.36–1.10 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 162.61, 162.58, 161.65 (d, $^{1}J_{C-F}$ = 243.2 Hz), 153.17, 149.05, 136.96, 136.38, 133.32, 131.96, 130.97 (d, $^{4}J_{C-F}$ = 2.7 Hz), 128.08 (d, $^{3}J_{C-F}$ = 7.8 Hz), 126.26, 121.63, 115.30 (d, $^{2}J_{C-F}$ = 21.3 Hz), 113.55, 111.81, 104.00, 103.96, 62.03, 51.83, 38.18, 29.27, 25.48, 25.02 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{28}\hbox {FN}_{6}\hbox {O}_{3}\hbox {S}$: 547.1922, found: 547.1920.

Methyl 7-((4-(4-chlorophenyl)thiazol-2-yl)amino)-8-(cyclohexyl(methyl)amino)-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-3-carboxylate (26)

Following general procedure F starting from 2-bromo-1-(4-chlorophenyl)ethanone (56 mg, 0.24 mmol) yielded a light-yellow powder (81 mg, 72 %); $\hbox {mp:}~ 261{-}263\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.95 (s, 1H), 9.59 (s, 1H), 9.16 (s, 1H), 8.56 (s, 1H), 8.16–8.11 (m, 3H), 7.52–7.49 (m, 3H), 3.85 (s, 3H), 2.82–2.74 (m, 1H), 2.70 (s, 3H), 1.91–1.87 (m, 2H), 1.73–1.69 (m, 2H), 1.55–1.51 (m, 1H), 1.36–1.04 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 162.65, 162.60, 153.18, 148.82, 136.90, 133.32, 133.21, 131.97, 131.92, 128.50, 127.78, 126.28, 121.63, 113.60, 111.82, 105.11, 104.02, 62.07, 51.83, 38.20, 29.24, 25.47, 25.01 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{28}\hbox {ClN}_{6}\hbox {O}_{3}\hbox {S}$: 563.1627, found: 563.1624.

Methyl 8-(cyclohexyl(methyl)amino)-7-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-3-carboxylate (27)

Following general procedure F starting from 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded a light-yellow powder (87 mg, 75 %); $\hbox {mp:}~ 257{-}258\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.68 (s, 1H), 9.52 (s, 1H), 9.16 (s, 1H), 8.42–8.39 (m, 1H), 8.25 (s, 1H), 8.14 (s, 1H), 8.01–7.95 (m, 2H), 7.90–7.86 (m, 1H), 7.64–7.54 (m, 3H), 7.20 (s, 1H), 3.83 (s, 3H), 2.89–2.81 (m, 1H), 2.73 (s, 3H), 1.92–1.88 (m, 2H), 1.75–1.71 (m, 2H), 1.57–1.53 (m, 1H), 1.40–1.06 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 162.66, 162.61, 152.90, 149.38, 136.69, 133.48, 133.32, 132.82, 132.02, 130.66, 128.27, 128.14, 127.39, 127.34, 126.34, 126.13, 125.82, 125.60, 125.49, 121.62, 113.93, 111.52, 108.12, 104.75, 61.99, 51.80, 37.66, 29.24, 25.47, 25.10 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{32}\hbox {H}_{31}\hbox {N}_{6}\hbox {O}_{3}\hbox {S}$: 579.2173, found: 579.2170.

General procedure G for the synthesis of compounds 28a–h and 50

To a stirred solution of ester intermediate (5a, 5c, 5d, 5e or 49, 5 mmol) in a mixed solvent of EtOH (25 mL) and $\hbox {H}_{2}\hbox {O}$ (5 mL), lithium hydroxide monohydrate (629 mg, 15 mmol) was added. The reaction mixture was stirred at $50\, ^{\circ }\hbox {C}$ for 1 h. After the reaction was complete (monitored by HPLC-MS analysis), a partial volume of the solvent was evaporated in vacuo, and the remaining mixture was acidified to pH 5–6 with 5 % hydrochloric acid. The formed precipitate was collected by filtration to give the corresponding crude carboxylic acid intermediate as a yellow solid. The intermediate (3.0 mmol) was dissolved in THF (50 mL), and then, HOBt (486 mg, 3.6 mmol) and EDCI (690 mg, 3.6 mmol) were added, followed by the addition of methylamine or dimethylamine (4.5 mmol, 2 M in THF) drop-wise. The reaction mixture was stirred at $60\, ^{\circ }\hbox {C}$. After the reaction was complete (monitored by HPLC-MS analysis), the solvent was evaporated carefully under reduced pressure to give a black oily residue, which was purified by silica gel column chromatography to give the amide intermediate (28 or 50).

3-(6-Amino-7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)-N-methylpropanamide (28a)

Following general procedure G starting from carboxylic acid type of 5a (1.03 g, 3.0 mmol) yielded a yellow powder (760 mg, 71 %); $\hbox {mp:}~ 125{-}127\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.91 (s, 1H), 7.78 (q, J = 4.5 Hz, 1H), 7.18 (s, 1H), 6.46 (s, 1H), 5.54 (s, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.74–2.67 (m, 1H), 2.58–2.56 (m, 6H), 2.47 (t, J = 7.2 Hz, 2H), 1.78–1.69 (m, 4H), 1.56–1.52 (m, 1H), 1.38–1.04 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{28}\hbox {N}_{5}\hbox {O}_{2}$: 358.2238, found: 358.2237.

3-(6-Amino-7-(cyclohexyl(methyl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)-N,N-dimethylpropanamide (28b)

Following general procedure G starting from carboxylic acid type of 5a (1.03 g, 3.0 mmol) yielded an orange powder (790 mg, 71 %); $\hbox {mp:}~ 190{-}192\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.90 (s, 1H), 7.18 (s, 1H), 6.46 (s, 1H), 5.53 (s, 2H), 3.02 (s, 3H), 2.88 (t, J = 7.2 Hz, 2H), 2.82 (s, 3H), 2.74–2.66 (m, 3H), 2.56 (s, 3H), 1.77–1.69 (m, 4H), 1.55–1.52 (m, 1H), 1.37–1.03 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{30}\hbox {N}_{5}\hbox {O}_{2}$: 372.2394, found: 372.2394.

3-(6-Amino-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)-N-methylpropanamide (28c)

Following general procedure G starting from carboxylic acid type of 5c (954 mg, 3.0 mmol) yielded a brown-yellow powder (705 mg, 71 %); $\hbox {mp:}~ 248-250\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.93 (s, 1H), 7.78 (q, J = 4.5 Hz, 1H), 7.13 (s, 1H), 6.48 (s, 1H), 5.62 (s, 2H), 3.77 (t, J = 4.2 Hz, 4H), 2.88 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 4.2 Hz, 4H), 2.57 (d, J = 4.5 Hz, 3H), 2.47 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{16}\hbox {H}_{22}\hbox {N}_{5}\hbox {O}_{3}$: 332.1717, found: 332.1715.

3-(6-Amino-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)-N,N-dimethylpropanamide (28d)

Following general procedure G starting from carboxylic acid type of 5c (954 mg, 3.0 mmol) yielded a tan powder (725 mg, 70 %); $\hbox {mp:}~ {>} 300\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 11.93 (s, 1H), 7.13 (s, 1H), 6.48 (s, 1H), 5.61 (s, 2H), 3.76 (t, J = 4.2 Hz, 4H), 3.03 (s, 3H), 2.89 (t, J = 7.2 Hz, 2H), 2.82 (s, 3H), 2.80 (t, J = 4.2 Hz, 4H), 2.68 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{3}$: 346.1874, found: 346.1868.

6-Amino-7-(cyclohexyl(methyl)amino)-N-methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (28e)

Following general procedure G starting from carboxylic acid type of 5d (948 mg, 3.0 mmol) yielded a dark-yellow powder (622 mg, 63 %); $\hbox {mp:}~ 277{-}278\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.53 (s, 1H), 9.37 (q, J = 4.8 Hz, 1H), 7.34 (s, 1H), 6.50 (s, 1H), 6.19 (s, 2H), 2.83 (d, J = 4.8 Hz, 3H), 2.76–2.70 (m, 1H), 2.58 (s, 3H), 1.80–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.39–1.06 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{24}\hbox {N}_{5}\hbox {O}_{2}$: 330.1924, found: 330.1922.

6-Amino-7-(cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (28f)

Following general procedure G starting from carboxylic acid type of 5d (948 mg, 3.0 mmol) yielded a yellow powder (731 mg, 71 %); $\hbox {mp:}~ 253{-}255\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.24 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 5.85 (s, 2H), 2.96 (s, 3H), 2.85 (s, 3H), 2.74–2.68 (m, 1H), 2.56 (s, 3H), 1.79–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.38–1.05 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{26}\hbox {N}_{5}\hbox {O}_{2}$: 344.2081, found: 344.2076.

6-Amino-N-methyl-7-(methyl(phenyl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (28g)

Following general procedure G starting from carboxylic acid type of 5e (930 mg, 3.0 mmol) yielded an orange powder (795 mg, 82 %); $\hbox {mp:}~ 270{-}272\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.57 (s, 1H), 9.20 (q, J = 4.8 Hz, 1H), 7.38 (s, 1H), 7.16 (t, J = 7.8 Hz, 2H), 6.71 (t, J = 7.2 Hz, 1H), 6.61 (d, J = 7.8 Hz, 2H), 6.60 (s, 1H), 6.27 (s, 2H), 3.14 (s, 3H), 2.81 ppm (d, J = 4.8 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{18}\hbox {N}_{5}\hbox {O}_{2}$: 324.1455, found: 324.1452.

6-Amino-N,N-dimethyl-7-(methyl(phenyl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (28h)

Following general procedure G starting from carboxylic acid type of 5e (930 mg, 3.0 mmol) yielded a yellow powder (728 mg, 72 %); $\hbox {mp:}~ 265{-}267\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.34 (s, 1H), 7.27 (s, 1H), 7.14 (t, J = 7.8 Hz, 2H), 6.69 (t, J = 7.2 Hz, 1H), 6.60–6.58 (m, 3H), 5.98 (s, 2H), 3.12 (s, 3H), 2.95 (s, 3H), 2.86 ppm (s, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{20}\hbox {N}_{5}\hbox {O}_{2}$: 338.1612, found: 338.1612.

3-(7-(Cyclohexyl(methyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl)-N-methylpropanamide (30a)

Following general procedure D starting from 28a (714 mg, 2.0 mmol) yielded a dark-yellow powder (607 mg, 73 % over two steps); $\hbox {mp:}~ {>} 300\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.18 (s, 1H), 9.10 (s, 1H), 8.16 (s, 1H), 7.92 (s, 2H), 7.80 (q, J = 4.5 Hz, 1H), 7.38 (s, 1H), 2.96 (t, J = 7.5 Hz, 2H), 2.72–2.67 (m, 1H), 2.63 (s, 3H), 2.57 (d, J = 4.5 Hz, 3H), 2.52 (t, J = 7.5 Hz, 2H), 1.80–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{29}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 417.2067, found: 417.2065.

3-(7-(Cyclohexyl(methyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl)-N,N-dimethylpropanamide (30b)

Following general procedure D starting from 28b (742 mg, 2.0 mmol) yielded a yellow powder (611 mg, 71 % over two steps); $\hbox {mp:}~ {>} 300\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.18 (s, 1H), 9.12 (s, 1H), 8.17 (s, 1H), 7.92 (s, 2H), 7.39 (s, 1H), 3.03 (s, 3H), 2.97 (t, J = 7.2 Hz, 2H), 2.82 (s, 3H), 2.75 (t, J = 7.2 Hz, 2H), 2.68–2.59 (m, 4H), 1.80–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.01 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{21}\hbox {H}_{31}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 431.2224, found: 431.2224.

N-methyl-3-(7-morpholino-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl)propanamide (30c)

Following general procedure D starting from 28c (662 mg, 2.0 mmol) yielded a yellow powder (585 mg, 75 % over two steps); $\hbox {mp:}~ 192{-}194\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.21 (s, 1H), 9.06 (s, 1H), 8.03 (s, 1H), 7.94 (br. s, 2H), 7.81 (q, J = 4.5 Hz, 1H), 7.34 (s, 1H), 3.81 (br. s, 4H), 2.97 (t, J = 7.2 Hz, 2H), 2.83 (br. s, 4H), 2.57 (d, J = 4.5 Hz, 3H), 2.52 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{17}\hbox {H}_{23}\hbox {N}_{6}\hbox {O}_{3}\hbox {S}$: 391.1547, found: 391.1541.

N,N-dimethyl-3-(7-morpholino-3-oxo-6-thioureido-3,4-dihydroquinoxalin-2-yl)propanamide (30d)

Following general procedure D starting from 28d (690 mg, 2.0 mmol) yielded a yellow powder (614 mg, 76 % over two steps); $\hbox {mp:}~ 184-186\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.21 (s, 1H), 9.06 (s, 1H), 8.03 (s, 1H), 7.93 (br. s, 2H), 7.35 (s, 1H), 3.80 (br. s, 4H), 3.04 (s, 3H), 2.97 (t, J = 7.2 Hz, 2H), 2.84 (br. s, 4H), 2.82 (s, 3H), 2.75 ppm (t, J = 7.2 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{25}\hbox {N}_{6}\hbox {O}_{3}\hbox {S}$: 405.1703, found: 405.1703.

7-(Cyclohexyl(methyl)amino)-N-methyl-3-oxo-6-thioureido-3,4-dihydroquinoxaline-2-carboxamide (30e)

Following general procedure D starting from 28e (658 mg, 2.0 mmol) yielded an orange powder (566 mg, 73 % over two steps); $\hbox {mp:}~ > 300\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.77 (s, 1H), 9.28 (s, 1H), 9.09 (q, J = 4.8 Hz, 1H), 8.45 (s, 1H), 8.12 (s, 2H), 7.59 (s, 1H), 2.82 (d, J = 4.8 Hz, 3H), 2.71–2.67 (m, 1H), 2.64 (s, 3H), 1.82–1.70 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{25}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 389.1754, found: 389.1748.

7-(Cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-6-thioureido-3,4-dihydroquinoxaline-2-carboxamide (30f)

Following general procedure D starting from 28f (686 mg, 2.0 mmol) yielded a yellow powder (659 mg, 82 % over two steps); $\hbox {mp:}~ 230{-}232\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.58 (br. s, 1H), 9.19 (s, 1H), 8.26 (s, 1H), 8.01 (s, 2H), 7.46 (s, 1H), 2.98 (s, 3H), 2.87 (s, 3H), 2.70–2.66 (m, 1H), 2.62 (s, 3H), 1.81–1.69 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.01 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{27}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 403.1911, found: 403.1907.

N-methyl-7-(methyl(phenyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxaline-2-carboxamide (30g)

Following general procedure D starting from 28g (646 mg, 2.0 mmol) yielded an orange powder (573 mg, 75 % over two steps); $\hbox {mp:}~ 240{-}242\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.85 (s, 1H), 9.34 (s, 1H), 8.96 (q, J = 4.8 Hz, 1H), 8.70 (s, 1H), 8.04 (s, 2H), 7.56 (s, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.78 (t, J = 7.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 2H), 3.14 (s, 3H), 2.80 ppm (d, J = 4.8 Hz, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{18}\hbox {H}_{19}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 383.1285, found: 383.1282.

N,N-dimethyl-7-(methyl(phenyl)amino)-3-oxo-6-thioureido-3,4-dihydroquinoxaline-2-carboxamide (30h)

Following general procedure D starting from 28h (674 mg, 2.0 mmol) yielded an orange powder (578 mg, 73 % over two steps); $\hbox {mp:}~ 184{-}186\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.73 (s, 1H), 9.27 (s, 1H), 8.62 (s, 1H), 7.96 (br. s, 2H), 7.49 (s, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.77 (t, J = 7.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 2H), 3.13 (s, 3H), 2.97 (s, 3H), 2.88 ppm (s, 3H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{21}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 397.1441, found: 397.1438.

3-(7-(Cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)-N-methylpropanamide (31)

Following general procedure E starting from 30a (83 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl) ethanone (52 mg, 0.24 mmol) yielded a light-yellow powder (69 mg, 65 %); $\hbox {mp:}~ 120{-}122\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.63 (s, 1H), 9.71 (s, 1H), 8.57 (s, 1H), 8.15 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.82 (q, J = 4.5 Hz, 1H), 7.50 (s, 1H), 7.45 (s, 1H), 7.27 (t, J = 9.0 Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 2.76–2.68 (m, 1H), 2.66 (s, 3H), 2.59 (d, J = 4.5 Hz, 3H), 2.54 (t, J = 7.8 Hz, 2H), 1.89–1.85 (m, 2H), 1.71–1.67 (m, 2H), 1.55–1.51 (m, 1H), 1.31–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 171.86, 162.29, 161.67 (d, $^{1}J_{C-F}$ = 243.2 Hz), 157.04, 155.13, 149.02, 138.67, 136.54, 130.93 (d, $^{4}J_{C-F}$ = 2.7 Hz), 129.84, 128.07 (d, $^{3}J_{C-F}$ = 8.1 Hz), 126.40, 122.68, 115.34 (d, $^{2}J_{C-F}$ = 21.2 Hz), 104.35, 101.39, 62.14, 38.65, 31.64, 29.23, 28.37, 25.52, 25.00, 24.97 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{32}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 535.2286, found: 535.2285.

3-(7-(Cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxalin-2-yl)-N,N-dimethylpropanamide (32)

Following general procedure E starting from 30b (86 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl) ethanone (52 mg, 0.24 mmol) yielded a yellow powder (89 mg, 81 %); $\hbox {mp:}~ 151{-}153\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.63 (s, 1H), 9.71 (s, 1H), 8.56 (s, 1H), 8.15 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.51 (s, 1H), 7.45 (s, 1H), 7.28 (t, J = 9.0 Hz, 2H), 3.05 (s, 3H), 2.98 (t, J = 7.2 Hz, 2H), 2.84 (s, 3H), 2.78–2.74 (m, 3H), 2.66 (s, 3H), 1.89–1.85 (m, 2H), 1.71–1.67 (m, 2H), 1.55–1.52 (m, 1H), 1.31–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{29}\hbox {H}_{34}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 549.2442, found: 549.2441.

3-(6-((4-(4-Fluorophenyl)thiazol-2-yl)amino)-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)-N-methylpropanamide (33)

Following general procedure E starting from 30c (78 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a flavo-green powder (65 mg, 64 %); $\hbox {mp:}~ 261{-}263\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.62 (s, 1H), 9.60 (s, 1H), 8.57 (s, 1H), 8.13 (dd, J = 8.7, 5.7 Hz, 2H), 7.82 (q, J = 4.5 Hz, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.27 (t, J = 9.0 Hz, 2H), 3.88 (br. s, 4H), 2.98 (t, J = 7.5 Hz, 2H), 2.86 (br. s, 4H), 2.59 (d, J = 4.5 Hz, 3H), 2.54 ppm (t, J = 7.5 Hz, 2H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{25}\hbox {H}_{26}\hbox {FN}_{6}\hbox {O}_{3}\hbox {S}$: 509.1766, found: 509.1768.

3-(6-((4-(4-Fluorophenyl)thiazol-2-yl)amino)-7-morpholino-3-oxo-3,4-dihydroquinoxalin-2-yl)-N,N-dimethylpropanamide (34)

Following general procedure E starting from 30d (81 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a yellow powder (74 mg, 71 %); $\hbox {mp:}~ 257{-}259\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.61 (s, 1H), 9.59 (s, 1H), 8.57 (s, 1H), 8.13 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.46 (s, 2H), 7.27 (t, J = 9.0 Hz, 2H), 3.88 (br. s, 4H), 3.05 (s, 3H), 2.98 (t, J = 7.2 Hz, 2H), 2.86 (br. s, 4H), 2.84 (s, 3H), 2.76 ppm (t, J = 7.2 Hz, 2H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 171.27, 162.46, 161.66 (d, $^{1}J_{C-F}$ = 243.2 Hz), 157.48, 155.13, 148.92, 137.45, 136.76, 130.94 (d, $^{4}J_{C-F}$ = 2.6 Hz), 129.52, 128.06 (d, $^{3}J_{C-F}$ = 8.0 Hz), 126.68, 119.28, 115.33 (d, $^{2}J_{C-F}$ = 21.2 Hz), 104.59, 102.31, 65.99, 52.39, 36.72, 34.89, 28.86, 27.94 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{26}\hbox {H}_{28}\hbox {FN}_{6}\hbox {O}_{3}\hbox {S}$: 523.1922, found: 523.1926.

7-(Cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-N-methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (35)

Following general procedure E starting from 30e (78 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded an orange powder (69 mg, 68 %); $\hbox {mp:}~ 252{-}254\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.23 (s, 1H), 9.97 (s, 1H), 9.20 (q, J = 4.8 Hz, 1H), 8.68 (s, 1H), 8.16 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.69 (s, 1H), 7.52 (s, 1H), 7.29 (t, J = 9.0 Hz, 2H), 2.85 (d, J = 4.8 Hz, 3H), 2.79–2.73(m, 1H), 2.68 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.32–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{26}\hbox {H}_{28}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 507.1973, found: 507.1972.

7-(Cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (36)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl) ethanone (52 mg, 0.24 mmol) yielded a dark-yellow powder (84 mg, 81 %); $\hbox {mp:}~ 163{-}165\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.02 (s, 1H), 9.85 (s, 1H), 8.65 (s, 1H), 8.16 (dd, J = 8.4 Hz, 5.7 Hz, 2H), 7.59 (s, 1H), 7.49 (s, 1H), 7.28 (t, J = 8.7 Hz, 2H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.66 (s, 3H), 1.90–1.86 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.29–0.98 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.24, 162.12, 161.71 (d, $^{1}J_{C-F}$ = 243.4 Hz), 153.22, 151.21, 149.04, 140.22, 137.36, 130.87 (d, $^{4}J_{C-F}$ = 2.6 Hz), 130.75, 128.09 (d, $^{3}J_{C-F}$ = 8.0 Hz), 126.14, 123.31, 115.37 (d, $^{2}J_{C-F}$ = 21.3 Hz), 104.87, 101.37, 62.07, 38.49, 36.94, 33.74, 29.16, 25.50, 24.99 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{30}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 521.2130, found: 521.2128.

N-methyl-7-(methyl(phenyl)amino)-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxamide (37)

Following general procedure E starting from 30g (76 mg, 0.2 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded an orange powder (79 mg, 82 %); $\hbox {mp:}~ 171{-}173\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.31 (s, 1H), 10.28 (s, 1H), 9.06 (q, J = 4.8 Hz, 1H), 8.95 (s, 1H), 8.14 (d, J = 7.5 Hz, 2H), 7.57 (s, 1H), 7.51–7.46 (m, 3H), 7.36 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.8 Hz, 2H), 6.76 (t, J = 7.5 Hz, 1H), 6.66 (d, J = 8.1 Hz, 2H), 3.17 (s, 3H), 2.82 ppm (d, J = 4.8 Hz, 3H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 163.27, 162.03, 154.99, 149.96, 149.47, 146.00, 141.81, 134.60, 134.17, 132.80, 128.75, 128.62, 128.17, 127.63, 126.84, 126.09, 117.89, 114.20, 105.77, 101.52, 39.43, 25.73 ppm; HRMS-ESI $m/z\, [M + \hbox {H}]^{+}$ calcd for $\hbox {C}_{26}\hbox {H}_{23}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 483.1598, found: 483.1597.

N,N-dimethyl-7-(methyl(phenyl)amino)-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxamide (38)

Following general procedure E starting from 30h (79 mg, 0.2 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded a dark-yellow powder (73 mg, 74 %); $\hbox {mp:}~ 165{-}167\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.17 (s, 1H), 10.19 (s, 1H), 8.94 (s, 1H), 8.13 (d, J = 7.5 Hz, 2H), 7.51–7.46 (m, 4H), 7.35 (t, J = 7.2 Hz, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.75 (t, J = 7.2 Hz, 1H), 6.66 (d, J = 8.1 Hz, 2H), 3.17 (s, 3H), 2.98 (s, 3H), 2.90 ppm (s, 3H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.06, 162.16, 153.34, 151.59, 149.89, 149.45, 141.08, 134.21, 133.92, 132.30, 128.76, 128.60, 127.60, 127.50, 126.57, 126.09, 117.74, 114.06, 105.46, 102.18, 36.92, 33.72 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{25}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 497.1754, found: 497.1752.

N-methyl-7-(methyl(phenyl)amino)-6-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (39)

Following general procedure E starting from 30g (76 mg, 0.2 mmol) and 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded a dark-yellow powder (78 mg, 73 %); $\hbox {mp:}~ 282{-}284\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.00 (s, 1H), 10.28 (s, 1H), 9.03 (q, J = 4.8 Hz, 1H), 8.75 (s, 1H), 8.41–8.38 (m, 1H), 8.03–7.94 (m, 3H), 7.64 (t, J = 7.5 Hz, 1H), 7.60–7.54 (m, 3H), 7.29 (s, 1H), 7.21 (t, J = 7.8 Hz, 2H), 6.77 (t, J = 7.2 Hz, 1H), 6.69 (d, J = 8.1 Hz, 2H), 3.20 (s, 3H), 2.79 ppm (d, J = 4.8 Hz, 3H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 163.22, 161.85, 154.78, 149.47, 149.09, 145.92, 141.95, 134.63, 133.52, 132.77, 132.74, 130.66, 128.77, 128.32, 128.18, 127.49, 126.85, 126.49, 125.87, 125.55, 125.46, 117.91, 114.23, 109.96, 101.53, 39.45, 25.70 ppm; HRMS-ESI $m/z\, [M + \hbox {H}]^{+}$ calcd for $\hbox {C}_{30}\hbox {H}_{25}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 533.1754, found: 533.1756.

N,N-dimethyl-7-(methyl(phenyl)amino)-6-((4-(naphthalen-1-yl)thiazol-2-yl)amino)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (40)

Following general procedure E starting from 30h (79 mg, 0.2 mmol) and 2-bromo-1-(naphthalen-1-yl)ethanone (60 mg, 0.24 mmol) yielded a dark-yellow powder (81 mg, 74 %); $\hbox {mp:}~ 162{-}164\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.85 (s, 1H), 10.19 (s, 1H), 8.73 (s, 1H), 8.42–8.39 (m, 1H), 8.03–7.93 (m, 3H), 7.64 (t, J = 7.5 Hz, 1H), 7.60–7.54 (m, 2H), 7.49 (s, 1H), 7.27 (s, 1H), 7.20 (t, J = 7.8 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 2H), 3.19 (s, 3H), 2.96 (s, 3H), 2.86 ppm (s, 3H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.03, 162.00, 153.10, 151.60, 149.43, 149.07, 141.20, 133.96, 133.51, 132.77, 132.28, 130.65, 128.77, 128.31, 128.15, 127.50, 127.47, 126.57, 126.48, 125.85, 125.54, 125.48, 117.76, 114.08, 109.64, 102.20, 39.41, 36.89, 33.70 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{31}\hbox {H}_{27}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 547.1911, found: 547.1908.

7-(Cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-6-((4-phenylthiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxamide (41)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-phenylethanone (48 mg, 0.24 mmol) yielded an orange powder (86 mg, 86 %); $\hbox {mp:}~ 165{-}167\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.02 (s, 1H), 9.84 (s, 1H), 8.65 (s, 1H), 8.11 (d, J = 7.2 Hz, 2H), 7.60 (s, 1H), 7.51 (s, 1H), 7.47 (t, J = 7.8 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 3.00 (s, 3H), 2.91 (s, 3H), 2.78–2.71 (m, 1H), 2.67 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.33–1.03 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.23, 161.96, 153.21, 151.18, 150.13, 140.22, 137.31, 134.21, 130.77, 128.58, 127.62, 126.11, 126.04, 123.28, 105.10, 101.33, 62.06, 38.46, 36.92, 33.73, 29.15, 25.49, 24.98 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{31}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 503.2224, found: 503.2223.

7-(Cyclohexyl(methyl)amino)-6-((4-(2-fluorophenyl)thiazol-2-yl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (42)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(2-fluorophenyl) ethanone (52 mg, 0.24 mmol) yielded a dark-yellow powder (76 mg, 73 %); $\hbox {mp:}~ 153{-}155\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.02 (s, 1H), 9.89 (s, 1H), 8.61 (s, 1H), 8.49–8.43 (m, 1H), 7.60 (s, 1H), 7.42–7.28 (m, 4H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.67 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.33–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.21, 161.40, 159.60 (d, $^{1}J_{C-F}$ = 247.4 Hz), 153.20, 151.27, 143.67, 140.14, 137.42, 130.70, 130.42 (d, $^{4}J_{C-F}$ = 2.4 Hz), 129.14 (d, $^{3}J_{C-F}$ = 8.4 Hz), 126.18, 124.83, 123.30, 121.74 (d, $^{3}J_{C-F}$ = 10.5 Hz), 115.80 (d, $^{2}J_{C-F}$ = 22.2 Hz), 109.91 (d, $^{2}J_{C-F}$ = 15.9 Hz), 101.41, 62.05, 38.40, 36.92, 33.73, 29.13, 25.49, 24.99 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{30}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 521.2129, found: 521.2131.

7-(Cyclohexyl(methyl)amino)-6-((4-(3-fluorophenyl)thiazol-2-yl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (43)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(3-fluorophenyl) ethanone (52 mg, 0.24 mmol) yielded a dark-yellow powder (88 mg, 85 %); $\hbox {mp:}~ 155{-}157\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.05 (s, 1H), 9.88 (s, 1H), 8.63 (s, 1H), 7.97–7.89 (m, 2H), 7.63 (s, 1H), 7.60 (s, 1H), 7.54–7.47 (m, 1H), 7.17 (td, J = 8.7 Hz, 2.1 Hz, 1H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.66 (s, 3H), 1.91–1.87 (m, 2H), 1.72–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.32–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{30}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 521.2129, found: 521.2130.

7-(Cyclohexyl(methyl)amino)-6-((4-(3,5-difluorophenyl)thiazol-2-yl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (44)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(3,5-difluorophenyl) ethanone (56 mg, 0.24 mmol) yielded a dark-yellow powder (87 mg, 81 %); $\hbox {mp:}~ 261{-}263\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.08 (s, 1H), 9.92 (s, 1H), 8.61 (s, 1H), 7.81 (dd, J = 8.7 Hz, 1.8 Hz, 2H), 7.75 (s, 1H), 7.60 (s, 1H), 7.20 (tt, J = 9.3 Hz, 1.8 Hz, 1H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.66 (s, 3H), 1.90–1.87 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.51 (m, 1H), 1.32–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.21, 162.80 (dd, $^{1,3}J_{C-F}$ = 243.4 Hz, 13.6 Hz), 162.31, 153.16, 151.34, 147.83, 140.01, 137.71 (t, $^{3}J_{C-F}$ = 10.2 Hz), 137.46, 130.67, 126.24, 123.28, 108.88 (dd, $^{2,4}J_{C-F}$ = 20.7 Hz, 5.2 Hz), 108.12, 102.78 (t, $^{2}J_{C-F}$ = 25.8 Hz), 101.58, 62.05, 38.40, 36.92, 33.73, 29.11, 25.49, 25.00 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{29}\hbox {F}_{2}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 539.2035, found: 539.2034.

6-((4-(3-Chloro-4-fluorophenyl)thiazol-2-yl)amino)-7-(cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (45)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(3-chloro-4-fluorophenyl)ethanone (60 mg, 0.24 mmol) yielded a dark-yellow powder (78 mg, 70 %); $\hbox {mp:}~ 164{-}166\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.03 (s, 1H), 9.89 (s, 1H), 8.60 (s, 1H), 8.24–7.97 (m, 2H), 7.64 (s, 1H), 7.59 (s, 1H), 7.49 (t, J = 9.0 Hz, 1H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.66 (s, 3H), 1.90–1.86 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.32–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.21, 162.35, 156.61 (d, $^{1}J_{C-F}$ = 245.8 Hz), 153.16, 151.30, 147.68, 140.08, 137.45, 132.16 (d, $^{4}J_{C-F}$ = 3.2 Hz), 130.68, 127.50, 126.82 (d, $^{3}J_{C-F}$ = 7.0 Hz), 126.22, 123.26, 119.85 (d, $^{2}J_{C-F}$ = 17.7 Hz), 117.00 (d, $^{2}J_{C-F}$ = 20.8 Hz), 106.41, 101.47, 62.03, 38.38, 36.92, 33.73, 29.12, 25.49, 25.00 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{29}\hbox {ClFN}_{6}\hbox {O}_{2}\hbox {S}$: 555.1740, found: 555.1741.

7-(Cyclohexyl(methyl)amino)-6-((4-(2,4-dichloro-5-fluorophenyl)thiazol-2-yl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (46)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(2,4-dichloro-5-fluorophenyl)ethanone (69 mg, 0.24 mmol) yielded a dark-yellow powder (102 mg, 87 %); $\hbox {mp:}~ 157{-}159\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.96 (s, 1H), 9.92 (s, 1H), 8.47 (s, 1H), 8.17 (d, J = 10.5 Hz, 1H), 7.93 (d, J = 6.9 Hz, 1H), 7.76 (s, 1H), 7.58 (s, 1H), 3.00 (s, 3H), 2.90 (s, 3H), 2.76–2.70 (m, 1H), 2.66 (s, 3H), 1.90–1.86 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.33–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.18, 161.46, 156.14 (d, $^{1}J_{C-F}$ = 245.2 Hz), 153.09, 151.39, 144.13, 139.95, 137.59, 133.15 (d, $^{3}J_{C-F}$ = 7.2 Hz), 131.76, 130.57, 126.30, 126.13 (d, $^{4}J_{C-F}$ = 2.6 Hz), 123.20, 119.14 (d, $^{2}J_{C-F}$ = 19.2 Hz), 118.70 (d, $^{2}J_{C-F}$ = 23.7 Hz), 112.18, 101.58, 61.98, 38.20, 36.91, 33.72, 29.06, 25.50, 25.02 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{28}\hbox {Cl}_{2}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 589.1350, found: 589.1349.

7-(Cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-6-((4-(2,4,6-trifluorophenyl)thiazol-2-yl)amino)-3,4-dihydroquinoxaline-2-carboxamide (47)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 2-bromo-1-(2,4,6-trifluorophenyl)ethanone (61 mg, 0.24 mmol) yielded a dark-yellow powder (90 mg, 81 %); $\hbox {mp:}~ 234{-}235\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 12.70 (s, 1H), 9.84 (s, 1H), 8.37 (s, 1H), 7.58 (s, 1H), 7.33 (t, J = 8.7 Hz, 2H), 7.29 (s, 1H), 2.98 (s, 3H), 2.88 (s, 3H), 2.78–2.71 (m, 1H), 2.66 (s, 3H), 1.90–1.86 (m, 2H), 1.72–1.68 (m, 2H), 1.56–1.52 (m, 1H), 1.33–1.03 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{27}\hbox {H}_{28}\hbox {F}_{3}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 557.1941, found: 557.1939.

6-((4-(4-Cyanophenyl)thiazol-2-yl)amino)-7-(cyclohexyl(methyl)amino)-N,N-dimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (48)

Following general procedure E starting from 30f (80 mg, 0.2 mmol) and 4-(2-bromoacetyl)benzonitrile (54 mg, 0.24 mmol) yielded an orange powder (80 mg, 76 %); $\hbox {mp:} 249{-}251\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 13.07 (s, 1H), 9.98 (s, 1H), 8.68 (s, 1H), 8.31 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 8.7 Hz, 2H), 7.81 (s, 1H), 7.60 (s, 1H), 3.00 (s, 3H), 2.91 (s, 3H), 2.77–2.70 (m, 1H), 2.66 (s, 3H), 1.91–1.87 (m, 2H), 1.71–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.32–1.02 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.20, 162.41, 153.21, 151.34, 148.27, 140.10, 138.33, 137.45, 132.60, 130.67, 126.64, 126.22, 123.35, 119.06, 109.67, 109.07, 101.57, 62.08, 38.51, 36.93, 33.73, 29.13, 25.50, 24.99 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{30}\hbox {N}_{7}\hbox {O}_{2}\hbox {S}$: 528.2176, found: 528.2175.

Ethyl 6-amino-7-(cyclohexyl(methyl)amino)-4-methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (49)

To a stirred solution of intermediate 5d (2.75 g, 8.0 mmol) in acetone (150 mL) was added $\hbox {K}_{2}\hbox {CO}_{3}$ (1.66 g, 12 mmol) and methyl iodide (0.60 mL, 9.6 mmol), and the mixture was stirred at $50\, ^{\circ }\hbox {C}$ for 6 h. The suspension was cooled and filtered. Then, the filtrate was concentrated under reduced pressure. The residue was dissolved in saturated ammonium chloride solution (100 mL) and extracted by $\hbox {CH}_{2}\hbox {Cl}_{2}$ (2 $\times$ 100 mL). The organic phases were combined, dried over anhydrous $\hbox {Na}_{2}\hbox {SO}_{4}$, and evaporated in vacuo to give a dark-yellow solid residue, which was purified by silica gel column chromatography ($\hbox {CH}_{2}\hbox {Cl}_{2}/\hbox {MeOH}$, 80:1) to give the title compound as a dark-yellow powder (2.21 g, 77 %); $\hbox {mp:}~ 187{-}188\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 7.32 (s, 1H), 6.62 (s, 1H), 6.20 (s, 2H), 4.28 (q, J = 7.2 Hz, 2H), 3.50 (s, 3H), 2.78–2.71 (m, 1H), 2.59 (s, 3H), 1.79–1.70 (m, 4H), 1.56–1.52 (m, 1H), 1.40–1.05 ppm (m, 8H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{27}\hbox {N}_{4}\hbox {O}_{3}$: 359.2078, found: 359.2077.

6-Amino-7-(cyclohexyl(methyl)amino)-N,N,4-trimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (50)

Following general procedure G starting from carboxylic acid type of 49 (991 mg, 3.0 mmol) yielded a yellow powder (890 mg, 83 %); $\hbox {mp:}~ 215{-}216\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 7.29 (s, 1H), 6.66 (s, 1H), 5.91 (s, 2H), 3.51 (s, 3H), 2.97 (s, 3H), 2.84 (s, 3H), 2.78–2.71 (m, 1H), 2.58 (s, 3H), 1.79–1.70 (m, 4H), 1.56–1.52 (m, 1H), 1.40–1.07 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{19}\hbox {H}_{28}\hbox {N}_{5}\hbox {O}_{2}$: 358.2238, found: 358.2238.

7-(Cyclohexyl(methyl)amino)-N,N,4-trimethyl-3-oxo-6-thioureido-3,4-dihydroquinoxaline-2-carboxamide (52)

Following general procedure D starting from 50 (714 mg, 2.0 mmol) yielded a dark-yellow powder (632 mg, 76 % over two steps); $\hbox {mp:}~ 192{-}194\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 9.32 (s, 1H), 8.76 (s, 1H), 8.15 (s, 2H), 7.56 (s, 1H), 3.58 (s, 3H), 2.99 (s, 3H), 2.87 (s, 3H), 2.73–2.67 (m, 1H), 2.65 (s, 3H), 1.83–1.70 (m, 4H), 1.56–1.53 (m, 1H), 1.36–1.02 ppm (m, 5H); HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{20}\hbox {H}_{29}\hbox {N}_{6}\hbox {O}_{2}\hbox {S}$: 417.2067, found: 417.2066.

7-(Cyclohexyl(methyl)amino)-6-((4-(4-fluorophenyl)thiazol-2-yl)amino)-N,N,4-trimethyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (53)

Following general procedure E starting from 52 (83 mg, 0.2 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone (52 mg, 0.24 mmol) yielded a dark-yellow powder (90 mg, 84 %); $\hbox {mp:}~ 223{-}224\, ^{\circ }\hbox {C}$; $^{1}\hbox {H}$ NMR (300 MHz, DMSO-d6): $\updelta$ = 10.02 (s, 1H), 8.97 (s, 1H), 8.03 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 7.65 (s, 1H), 7.51 (s, 1H), 7.31 (t, J = 8.7 Hz, 2H), 3.73 (s, 3H), 3.01 (s, 3H), 2.89 (s, 3H), 2.81–2.74 (m, 1H), 2.68 (s, 3H), 1.92–1.88 (m, 2H), 1.72–1.68 (m, 2H), 1.55–1.52 (m, 1H), 1.34–1.03 ppm (m, 5H); $^{13}$C NMR (150 MHz, DMSO-d6): $\updelta$ = 165.16, 162.33, 161.66 (d, $^{1}J_{C-F}$ = 243.4 Hz), 152.38, 149.76, 148.59, 140.46, 137.27, 131.78, 130.96, 127.51 (d, $^{3}J_{C-F}$ = 8.0 Hz), 126.46, 124.05, 115.63 (d, $^{2}J_{C-F}$ = 21.4 Hz), 105.12, 101.15, 61.94, 38.36, 36.94, 33.72, 29.10, 28.92, 25.50, 24.99 ppm; HRMS-ESI $m/z\, [\hbox {M}+\hbox {H}]^{+}$ calcd for $\hbox {C}_{28}\hbox {H}_{32}\hbox {FN}_{6}\hbox {O}_{2}\hbox {S}$: 535.2286, found: 535.2286.

Biology

HCV antiviral assay The Huh7-ET (luc-ubi-neo/ET) cell line which harbors a dicistronic self-replicating HCV RNA replicon with a firefly luciferase gene was used for antiviral evaluation [51]. The activity of the luciferase reporter is directly proportional to HCV RNA levels. Briefly, the ET cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 2 mM L-glutamine, nonessential amino acids, 10 % fetal bovine serum (FBS), and 0.25 mg/mL G418 (Invitrogen, Carlsbad, CA). The cells were seeded in two identical sets of 96-well plates used for analysis of antiviral activity or cell numbers (cytotoxicity) at a density of 5000/well in $100 \,\upmu \hbox {L}$ of DMEM without G418 overnight. Serial dilutions of compounds solubilized in DMSO were prepared in DMEM and then added to the appropriate wells. After 72 h of incubation, one set of the cells was processed to assess the replicon-derived luciferase activity with the Steady-Glo luciferase assay system (Promega, Madison, WI) according to manufacturer’s instruction. Another set of the plates was used to determine cytotoxic effect of the compounds using a tetrazolium-based CytoTox-1 cell proliferation assay (Promega, Madison WI). Each data point represents an average of four replicates to derive $\hbox {EC}_{50}$ (concentration of compound to inhibit HCV RNA replication activity by 50 %), $\hbox {CC}_{50}$ (concentration of compound to decrease cell viability by 50 %), and $\hbox {SI}_{50}$ (selective index calculated as the $\hbox {CC}_{50}/\hbox {EC}_{50}$ ratio) values.