Discovery and biological evaluation of Phthalazines as novel non-kinase TGFβ pathway inhibitors

Abstract

TGFβ is crucial for the homeostasis of epithelial and neural tissues, wound repair, and regulating immune responses. Its dysregulation is associated with a vast number of diseases, of which modifying the tumor microenvironment is one of vital clinical interest. Despite various attempts, there is still no FDA-approved therapy to inhibit the TGFβ pathway. Major mainstream approaches involve impairment of the TGFβ pathway via inhibition of the TGFβRI kinase. With the purpose to identify non-receptor kinase-based inhibitors to impair TGFβ signaling, an in-house chemical library was enriched, through a computational study, to eliminate TGFβRI kinase activity. Selected compounds were screened against a cell line engineered with a firefly luciferase gene under TGFβ-Smad-dependent transcriptional control. Results indicated moderate potency for a molecule with phthalazine core against TGFβ-Smad signaling. A series of phthalazine compounds were synthesized and evaluated for potency. The most promising compound (10p) exhibited an IC₅₀ of 0.11 ± 0.02 µM and was confirmed to be non-cytotoxic up to 12 µM, with a selectivity index of approximately 112-fold. Simultaneously, 10p was confirmed to reduce the Smad phosphorylation using western blot without exhibiting inhibition on the TGFβRI enzyme. This study identified a novel small-molecule scaffold that targets the TGFβ pathway via a non-receptor-kinase mechanism.

Graphical Abstract

Transforming growth factor-beta (TGFβ) is a profibrogenic cytokine, which plays a crucial role in metazoan biology. Nearly all the cell types respond to TGFβ, it maintains homeostasis of epithelial and neural tissues, aids in wound repair, and regulates aspects of the immune system.[1] The release of TGFβ stimulates the TGFβ receptors (TGFβRI and TGFβRII), which activates SMAD proteins that modulate gene expression (Figure 1).

Figure 1:

TGFβ signaling pathway via SMAD-TGFβ ligand binds to transmembrane TGFβ type-II receptor (TGFβRII) and induces its phosphorylation, followed by autophosphorylation of TGFβ type-I (TGFβRI) receptor forming a heterotetrameric receptor complex with two type I receptors and two type II receptors. Activated TGFβRI initiates TGFβ/Smad signaling by phosphorylating receptor-associated Smads (R-Smads), Smad2, and Smad3. The activated R-Smads form a complex with Co-Smad and shuttles into the nucleus, binding to DNA at Smad-binding element in the promoter region of the TGFβ targeted gene. TGFβ receptor complex can also activate non-smad dependent pathways like Ras/ERK pathway, PI3K/AKT pathway, etc.

Perturbation of the TGFβ pathway can interrupt immune-modulatory activities, which has implications in many diseases like cancer, asthma, nephritis, cardiovascular disease, rheumatoid arthritis, and multiple sclerosis.[2–5] In the context of cancer, TGFβ exhibits a functional switch from a tumor suppressor to a tumor promoter, which is known as the “TGFβ paradox.”[6] In a metastatic state, the concentration of TGFβ increases significantly in the tumor microenvironment.[7,8] TGFβ is responsible for the polarization of M1 macrophages to tumor-associated macrophages(TAM).[9] Studies involving obstruction of TGFβ signaling exhibit an improvement in the CD8⁺ T-cell mediated immune response, an increase in cytotoxic T-cells and NK cells at metastatic sites, and suppression of T-regs.[10–12] Further studies revealed that upregulation of TGFβRII in the tumor microenvironment correlates to drug resistance to chemotherapeutics like cisplatin and targeted therapeutics such as Erlotinib, Gefitinib, Crizotinib, Vemurafenib, and Sorafenib.[13] Suppressing TGFβ has also been shown to enhance the efficacy of PD-1 checkpoint inhibitors.[14] Owing to its role in immune regulation, TGFβ is a highly researched cytokine in cancer, autoimmune diseases, and infectious disease.[15–22]

In the past few decades, modifying TGFβ signaling has been investigated with TGFβRI kinase inhibitors, such as 1 (LY-2157299), 2 (SB-505124), 3 (SD-208), and 4 (GW6604) (Figure 2), and more. [23–38] These inhibitors are ATP mimetic drugs and are designed to target TGFβRI directly. However, like other kinase inhibitors, they have the potential for promiscuity within the kinome as the ATP pocket is heavily conserved.[39–42] Therefore, targeting TGFβRI possesses off-target liabilities by inadvertently blocking kinases similar to TGFβRI, such as ALK-4 and ALK-7.[24,43] Moreover, TGFβ can also activate numerous non-canonical pathways, which can play an essential role in tumor suppression, and kinase inhibition can interfere with the activation of non-canonical TGFβ pathways generating undesired side effects.[44–47] Therefore, there is a need to investigate therapeutic strategies that impair the TGFβ-Smad pathway without directly interfering with kinase activity. A similar approach has resulted in the characterization of SIS3, SMAD3 specific inhibitors, Asiaticoside, a SMAD 7 modulator, and Pirfenidone, which can decrease TGFβ protein expression.[20,48–49] In this study, we explored further to identify non-receptor kinase-based inhibitors to impair TGFβ signaling.

Figure 2:

Small molecule inhibitors of TGFβRI

In order to develop a new approach to target the TGFβ pathway, we performed a phenotypic screening using HEK293 cell-line, with a luciferase reporter under transcriptional regulation by TGFβ. Using the luciferase assay, we screened a diverse, in-house library that was computationally predicted to have limited-to-no TGFβRI inhibition. Hits from the designed experiment were confirmed not to have direct TGFβRI inhibition using an enzymatic inhibitory assay (Figure 3). From this approach, we uncovered a novel phthalazine chemotype that exhibited potent impairment of the TGFβ pathway without any direct inhibition of the TGFβRI kinase. We extensively explored the chemotype by generating a small, focused phthalazine library and evaluated the library in cell and enzymatic assays. This study identified 10p as the most efficacious inhibitor of the TGFβ pathway with minimal cytotoxicity and no inhibition of the TGFβRI kinase. Additionally, inhibition of Smad phosphorylation was confirmed using western blot.

Figure 3:

Identification of non-receptor-kinase TGFβ pathway inhibitors

RESULTS AND DISCUSSION

Chemistry.

Compounds 8a-r and 10a-r were synthesized according to the synthetic route depicted in Scheme 1. Isoindoline-1,3-dione 5 was reacted with hydrazine hydrate in ethanol to afford 2,3-dihydrophthalazine-1,4-dione, which was subsequently reacted with phosphorus oxychloride (POCl₃) to give 1,4-dichlorophthalazine 6.[50] 1,4-dichlorophthalazine was coupled to an amine in ethanol to yield 7 (or 9a-r), which was further reacted with a benzyl substituted piperazine to give 8a-r (or 10a-r). To generate a carbon-carbon bond with the phthalazine core, 7 underwent Suzuki-Miyaura reaction with a respective boronic acid pinacolate ester to generate final compounds 11 and 12, as shown in Scheme 2. [51]

Scheme 1:

Synthesis of 8a-r and 10a-r: (a.i) NH₂NH₂, EtOH, reflux for 12h; (a.ii) POCl₃, DMF, 120°C, 2h; (b) EtOH, 65°C, 3h; (c) n-BuOH, overnight, 150°C.

Scheme 2:

Synthesis of 11 and 12: (a) Pd(PPh₃)4, Cs₂CO₃, DMF:H₂O (4:1), 115°C, MW, 1h.

To understand the importance of both the nitrogen, in the core, on potency against the TGFβ pathway, compounds were generated with an isoquinoline core in place of the phthalazine. To synthesize isoquinolines, 4-bromoisoquinoline was oxidized using m-chloroperbenzoic acid, which was further chlorinated using POCl₃ to give 14.[52] 1-(3-(trifluoromethyl)benzyl)piperazine was then coupled to intermediate 14 to yield 15, which were subjected to Buchwald-Hartwig amination with 4-morpholinoaniline to give final compound 16. To generate the isoquinoline isomer, 4-morpholinoaniline was reacted with 14 and then subjected to a Buchwald–Hartwig cross-coupling reaction to generate 18 (Scheme 3).[53]

Scheme 3:

Synthesis of 16 and 18: (a.i)m-CPBA, DCM, rt, 3h; (a.ii) POCI₃, DMF, 120°C, 2h; (b) n-BuOH, overnight, 150°C; (c) Pd₂dba₃, (±)-BINAP, Dioxane, K^tBuO, 110°C, overnight; (d) EtOH, 65°C, 3h.

Phenotypical Screening.

To identify non-kinase inhibitor scaffolds that could potentially block the TGFβ pathway, we performed a computational study using our in-house library to identify scaffolds that possessed a low potential for kinase inhibition (Supplementary section 7). Computationally filtered compounds were screened using HEK293 cell line with a luciferase reporter, under transcriptional regulation by TGFβ. This cell line was engineered to monitor input into the TGFβ-Smad signaling pathway by placing the firefly luciferase gene under Smad-dependent transcriptional control. Therefore, a decrease in luminescent signal correlates to a reduction in TGFβ signaling. We were interested in identifying strategies to impair the TGFβ pathway without directly inhibiting kinase potency in an attempt to limit toxicity and improve efficacy while targeting the TGFβ pathway.

From the initial screen, we identified a phthalazin-1-amine (8a) with potency against the TGFβ pathway with an IC₅₀ of 10.04 ± 0.62 µM as refereed by a reduction in luminescent signal. This impairment of the TGFβ pathway could stem from few significant reasons. One, 8a could possess TGFβRI inhibitory activity, directly blocking the Smad-mediated activation of the firefly luciferase gene. Two, 8a could be cytotoxic and non-specifically impair cell viability, causing a reduction in cellular processes such as gene transcription and protein synthesis. Three, interaction with luciferase reporter system a cross screening was performed in HEK293 cell line with different pathways to confirm no inhibition against another pathway, to eliminate the possibility of luciferase reporter system interference (Supplementary section 8.3). On eliminating the above three possibilities, we would support that the compounds interfere with TGFβ signaling via a novel mechanism. To confirm this hypothesis, we performed immunoblot analysis to observe reduced Smad2/3 phosphorylation when treated with an inhibitor.

To eliminate the possibility of cytotoxicity, we incorporated matched viability assays to determine a preliminary safety window. When running the reporter assay, we assessed TGFβ pathway impairment (IC₅₀) as well as cell viability (GI₅₀) to establish a Selectivity Index (GI₅₀/IC₅₀). Using this approach, we determined compound 8a had a selectivity index of 4, indicating that at non-cytotoxic concentrations of 45.62 ± 2.29 µM, the compound was impairing the TGFβ pathway. Next, we subjected 8a to a biochemical inhibitor assay to assess direct inhibition of the TGFβRI kinase, and 8a did not exhibit TGFβRI inhibition (Table 4, Entry 1). Considering these results, 8a appeared to block the TGFβ pathway without inhibiting TGFβRI and was pursued further as a hit candidate. To explore structure-activity relationships (SAR) further, structure 8a was classified into three regions (Figure 4). Substitution on the benzylic ring (Region A) of 8a was investigated as depicted in Table 1 (Entries 1–18). Introduction of bromo at the meta-position (8c, IC₅₀=1.32±0.23 µM; GI₅₀=13.14±0.54 µM; Selectivity Index=9.98) improved the selectivity index compared to its positional isomers 8b and 8d. Even though the replacement of m-bromo with m-fluoro (8e) or m-chloro (8f) did not exhibit improvement in potency, inhibition improved as the size of the halogen increased. Further SAR studies focused on modifying the meta position with electron-withdrawing (8g-j) or donating (8k-m) groups. Electron withdrawing groups exhibited high selectivity for targeting the TGFβ pathway compared to cell viability, and the highest selectivity index was observed with 8g (IC₅₀=0.24±0.03 µM, GI₅₀=16.85±0.25 µM, Selectivity Index =69.59).

Table 4:

Inhibition of TGFβRI at 2.0 µM, and 20 µM.

Compd	Percent Inhibition at 2.0 µM	Percent Inhibition at 2.0 µM
8a	0%	0%
8g	0%	0%
10p	0%	0%
10q	0%	0%
LY-2157299	73%	100%

Figure 4:

SAR study of the phthalazine scaffold.

Table 1:

TGFβ inhibitory potency and cytotoxicity of compounds 8a-r

Compd	R	TGFβ pathway impairment IC50 (µM)	HEK293 viability GI50 (µM)	Selectivity index (GI50/IC50)
8a	H	10.04±0.62	45.62±2.29	4.54
8b	o-Br	5.84±0.36	12.4±0.21	2.12
8c	m-Br	1.32±0.23	13.14±0.54	9.98
8d	p-Br	11.53±0.08	18.49±1.16	1.60
8e	m-F	7.37±0.26	35.31±1.59	4.79
8f	m-Cl	1.56±0.22	14.13±1.48	9.05
8g	m-CF 3	0.24±0.03	16.85±0.25	69.59
8h	m-NO 2	1.23±0.10	9.26±0.32	7.55
8i	m-CN	1.72±0.11	14.64±0.53	8.51
8j	m-COOMe	0.17±0.02	7.59±0.29	44.65
8k	m-OMe	5.27±0.69	10.00±0.66	1.89
8l	m-OCF3	5.89±0.74	98.29±0.5	16.69
8m	m-Me	1.96±0.44	13.48±1.15	6.87
8n	m,p-di-Cl	5.47±0.32	36.24±3.3	6.62
8o	m,p-di-F	4.00±0.85	12.26±0.43	3.06
8p	o,o-di-Cl	9.45±0.28	14.21±0.54	1.50
8q	o,p-di-F	3.82±0.99	11.50±0.26	3.01
8r	m-Cl,p-F	7.49±0.39	11.48±2.49	1.53

We then explored changing the flexible carbon-nitrogen linker to a ridged carbon-carbon linker, as shown in Table 2 (Entries 11–12). Carbon-carbon bonds were generated with benzene (11) or pyrazole (12), and we observed a loss of potency by 12-fold and 33-fold compared to 8g, respectively. We then modified the phthalazine at Region B ring system to assess the significance of the phthalazine-core (Table 2 (Entries 16, 18)). When the nitrogen adjacent to morpholinoaniline was removed, an 8-fold reduction in potency was observed (16, IC₅₀=1.97±0.21 µM; GI₅₀=11.24±1.14 µM; Selectivity Index =5.71). Whereas, removal of the other nitrogen caused a complete loss of inhibition (18, IC₅₀=20.10±0.73 µM; GI₅₀=49.52±2.35 µM; Selectivity Index =2.46), which explicitly indicates the phthalazine-core is integral for potency.

Table 2:

TGFβ inhibitory potency and cytotoxicity of compounds 11, 12, 16, and 18 in HEK-293 cell line

Compd	Structure	TGFβ pathway impairment IC50 (µM)	HEK293 viability GI50 (µM)	Selectivity index (GI50/IC50)
11		3.01±0.99	11.14±0.88	3.71
12		8.17±0.67	20.55±3.24	2.51
16		1.97±0.21	11.24±1.14	5.71
18		20.10±0.73	49.52±2.35	2.46

We progressed 8g as a lead candidate and further explored the SAR on this scaffold by modifying the phthalazine substitution at Region C (Figure 4), depicted in Table 3(Entries 10a-r). Introduction of saturated acyclic amines (10a-b), cyclic amines (10c-f), or a combination of both (10g-i) exhibited no improvement, as compounds were either less potent or more cytotoxic. By replacing saturated amines with aromatic amines improved potency and selectivity profiles (Table 3, Entries 10j-10o). Which was further improved by increasing the size of the substitution on the aromatic ring. Further introduction of mono and di-methyl substitution on the morpholine ring improved the potency and selectivity (10p-r). Overall, SAR at this position suggested bulky, para substitution on aromatic amines is most suitable for impairing the TGFβ pathway with limited cytotoxicity was observed with 10p (IC₅₀= 0.11±0.02µM, GI₅₀=12.32±1.02µM, Selectivity Index =112.0).

Table 3:

TGFβ inhibitory potency and cytotoxicity of compounds 10a-r

Compd	-NH-R’	TGFβ pathway impairment IC50 (μM)	HEK293 viability GI50 (μM)	Selectivity index (GI50/IC50)
10a		19.05±0.14	73.58±3.24	3.86
10b		7.12±0.19	12.58±2.75	1.77
10c		5.91±0.79	38.41±2.7	6.49
10d		7.55±0.67	12.14±0.65	1.61
10e		3.91±0.67	10.26±0.25	2.63
10f		7.51±0.72	72.28±3.63	9.62
10g		13.78±1.00	76.41±0.56	5.54
10h		17.81±0.75	110.15±6.29	6.18
10i		7.08±0.32	8.14±0.04	1.15
10j		7.16±0.68	10.39±0.12	1.45
10k		2.36±0.315	53.81±0.17	22.81
10l		4.54±0.28	40.07±1.37	8.83
10m		0.88±0.06	16.64±0.08	18.82
10n		0.36±0.03	13.39±1.25	37.38
10o		0.47±0.12	18.01±0.29	38.09
10p		0.11±0.02	12.32±1.02	112.0
10q		0.19±0.01	20.01±0.30	105.31
10r		0.21±0.01	9.69±0.14	46.14

Since our primary aim was to investigate TGFβ-Smad pathway inhibitors without directly interfering with the receptor, we performed an enzymatic assay to confirm that compounds have limited to no inhibition at the TGFβRI kinase. We performed single point inhibition in an enzymatic assay for compounds 8a, 8c, 8g, 10m, 10n, and 10o at 2.0 µM, and 20 µM. We observed no inhibition for the compounds at both 2.0 µM, and 20 µM, where the control compound (LY-2157299) showed 73% and 100% inhibition, respectively, of TGFβRI (Table 4 and Supplementary Section 9). These results suggest that these compounds inhibit TGFβ-Smad signaling via a non-receptor-kinase mechanism. Next, western blot analysis was performed to confirm TGFβ pathway inhibition by compounds 8g and 10p; inhibition of phosphorylation of Smad2/3 in isolated CD4⁺ T cell was observed by performing immunoblot analysis at concentrations of 0.5 and 5 µM (Figure 5). Additionally, a selectivity enzymatic screening panel was also performed against few kinases like CSF-1R, TRKA, FLT3, AURK-A, Nek-2 with compounds 8g, 10n, and 10p; we observed almost no inhibition against any of these kinases showing their selectivity for TGFβ-Smad pathway and also resonating with the finding that compounds are non-kinase inhibitors. (Supplementary section 10).

Figure 5:

Western blot to study the inhibition of smad-phosphorylation by designed inhibitors 8g and 10p was observed in CD4⁺ T cells from wild-type mice spleens which were treated with human TGFβ1 (and without for negative control)

CONCLUSIONS

The TGFβ pathway is essential for immune regulation, which has implications in cancer, autoimmune disease, and infectious disease. However, there is not an FDA-approved therapy to target the TGFβ pathway. Because most strategies targeting the TGFβ pathway have relied on direct inhibition of TGFβRI, we attempted to identify non-receptor-based methods to impair TGFβ signaling. By completing a phenotypic screen using a cell line engineered with a firefly luciferase gene under Smad-dependent transcriptional control, we identified a novel phthalazine scaffold that blocked TGFβ signaling without directly inhibiting TGFβRI. By completing SAR exploration, we identified compound 10p, which inhibited the TGFβ pathway at an IC₅₀ of 0.11±0.02µM. Compound 10p did not display cytotoxic effects until around 12 µM, and exhibited a selectivity index of approximately 112 fold. Significantly, 10p reduced the Smad2/3 phosphorylation without inhibiting the TGFβRI enzyme directly. Therefore, we discovered a novel small-molecule scaffold that targets the TGFβ pathway through a non-receptor-kinase mechanism. Work is being completed to identify the mechanism of action of the phthalazine scaffold, which may uncover novel, druggable pathways within the TGFβ signaling cascade.

EXPERIMENTAL SECTION

Compounds 6, 7, 8a-r, 9a-r, 10a-r, and 11–18 were prepared according to the mentioned schemes. Synthesis and characterization are presented in the Supporting Information. The purity of all compounds was determined to be ≥95% by HPLC and LCMS system at wavelengths of 214 and 254 nm.

Synthesis of 4-(4-benzylpiperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8a)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-benzylpiperazine (30.8mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Greenish yellow solid; Yield: 28%; R_f = 0.39 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.93 (m, 2H, Ar-H), 7.78 – 7.71 (m, 2H, Ar-H), 7.49 (d, J = 8.3 Hz, 2H), 7.40–7.30 (m, 4H, Ar-H), 7.27 (d, J = 7.1 Hz, 1H), 6.90 (d, J = 9.0 Hz, 2H), 3.85 (t, J = 4.0 Hz, 4H), 3.64 (s, 2H), 3.37 (brs, 4H), 3.10 (t, J = 4.0 Hz, 4H), 2.72 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.36, 137.61, 131.05, 129.29, 128.28, 127.20, 125.14, 123.60, 122.08, 116.83, 66.95, 63.09, 53.05, 51.10, 50.12; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₂N₆O 481.37, found 481.38.

Synthesis of 4-(4-(2-bromobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8b)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and added 1-(2-bromobenzyl)piperazine (44.6mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Pale yellow solid; Yield: 57%; R_f = 0.47 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.06 – 7.96 (m, 2H, Ar-H), 7.79 – 7.72 (m, 2H, Ar-H), 7.54 (dd, J = 8.0, 1.1 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.29 (td, J = 7.5, 1.2 Hz, 1H), 7.11 (td, J = 7.7, 1.7 Hz, 1H), 6.90 (d, J = 9.0 Hz, 2H), 3.85 (t, J = 4.0 Hz, 4H),) 3.73 (s, 2H), 3.37 (brs, 4H), 3.10 (t, J = 4.0 Hz, 4H), 2.78 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.38, 137.36, 132.70, 131.08, 130.81, 128.43, 127.29, 125.16, 124.60, 123.63, 122.09, 116.84, 66.95, 61.74, 53.17, 51.29, 50.11; LCMS (ESI): Calculated for [M]⁺ C₂₉H₃₁BrN₆O 559.51, found 559.60.

Synthesis of 4-(4-(3-bromobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8c)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-bromobenzyl)piperazine (44.6mg, 0.175mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 49%; R_f = 0.456 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J = 6.3 Hz, 1H), 7.95 (brs, 1H, NH), 7.73 (dd, J = 6.2, 2.7 Hz, 2H), 7.52 (s, 2H), 7.49 (s, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 6.88 (dd, J = 8.9, 2.9 Hz, 2H), 3.84 (brs, 4H), 3.57 (brs, 2H), 3.35 (brs, 4H), 3.08 (brs, 4H), 2.68 (brs, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 155.52, 149.81, 149.78, 147.26, 140.54, 132.01, 131.00, 130.96, 130.20, 129.84, 127.70, 125.06, 123.53, 122.41, 122.01, 116.79, 66.95, 62.45, 53.14, 51.21, 50.12; LCMS (ESI): Calculated for [M+2] C₂₉H₃₁BrN₆O 561.53, found 561.35.

Synthesis of 4-(4-(4-bromobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8d)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(4-bromobenzyl)piperazine (44.6mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 65%; R_f = 0.42 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.02 – 7.94 (m, 2H, Ar-H), 7.77 – 7.70 (m, 2H, Ar-H), 7.50 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 3.84 (t, J = 4.0 Hz, 4H), 3.55 (s, 2H), 3.34 (brs, 4H), 3.08 (t, J = 4.0 Hz, 4H), 2.67 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.28, 137.04, 131.35, 131.04, 130.98, 130.81, 125.05, 123.53, 122.04, 120.89, 116.78, 66.94, 62.35, 53.10, 51.20, 50.11; LCMS (ESI): Calculated for [M]⁺ C₂₉H₃₁BrN₆O 559.51, found 559.66.

Synthesis of 4-(4-(3-fluorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8e)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-fluorobenzyl)piperazine (34mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Greenish yellow solid; Yield: 25%; R_f = 0.44 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, DMSO) δ 9.06 (d, J = 7.8 Hz, 1H), 8.93 (brs, 1H, NH), 8.17 (d, J = 7.8 Hz, 1H), 8.10 (p, J = 8.5 Hz, 2H), 7.65 (d, J = 10.1 Hz, 1H), 7.55–7.45 (m, 2H, Ar-H), 7.35 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 9.0 Hz, 2H), 4.42 (s, 2H), 3.72 (t, J = 4.0 Hz, 4H), 3.55 – 3.44 (m, 4H), 3.37 (brs, 4H), 3.20 – 2.99 (m, 4H); ¹³C NMR (101 MHz, DMSO) δ 163.54, 161.11, 153.86, 150.44, 135.70, 132.64, 131.24, 126.91, 128.26, 126.91, 126.40, 126.12, 123.55, 118.80, 116.85, 116.39, 66.46, 58.24, 50.73, 48.72, 47.68; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₁FN₆O 499.26, found 499.42.

Synthesis of 4-(4-(3-chlorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8f)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-chlorobenzyl)piperazine (36.7mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Pale yellow solid; Yield: 36%; R_f = 0.46 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.04 – 7.98 (m, 2H, Ar-H), 7.80 – 7.73 (m, 2H, Ar-H), 7.50 (d, J = 8.5 Hz, 2H), 7.37 (s, 1H, NH), 7.25 (s, 3H), 6.91 (d, J = 9.0 Hz, 2H), 3.86 (t, J = 4.0 Hz, 4H), 3.59 (s, 2H), 3.37 (brs, 4H), 3.11(t, J = 4.0 Hz, 4H) 2.70 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.52, 147.38, 147.17, 140.03, 138.68, 134.16, 131.20, 131.17, 129.54, 129.15, 127.34, 127.26, 125.13, 123.63, 122.24, 116.84, 66.94, 62.46, 53.06, 51.12, 50.07; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₁ClN₆O 515.23, found 515.46.

Synthesis of N-(4-morpholinophenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (8g)

4-chloro- N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (42.7mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 65%; R_f = 0.42 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05 – 7.98 (m, 2H, Ar-H), 7.79 – 7.73 (m, 2H, Ar-H), 7.62 (s,1H, NH), 7.58 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 3H), 7.44 (t, J = 7.6 Hz, 1H), 6.90 (d, J = 8.9 Hz, 2H), 3.85 (t, J = 4.0 Hz, 4H), 3.66 (s, 2H), 3.37 (brs, 4H), 3.10 (t, J = 4.0 Hz, 4H), 2.71 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.49, 139.11, 132.39, 131.18, 131.15, 130.76, 130.44, 128.74, 125.68, 125.11, 124.01, 123.63, 122.83, 122.21, 116.82, 66.94, 62.52, 53.11, 51.16, 50.07; IR Absorption ν_max/cm⁻¹ 3314.72 (NH), 2961.11, 2920.26, 2849.86 (Ar-H), 1325.63 (C-N aromatic), 1112.64 (C-O aliphatic ether), 1070.03 (C-N aliphatic) ; LCMS (ESI): Calculated for [M+1]⁺ C₃₀H₃₁F₃N₆O 549.25, found 549.50.

Synthesis of N-(4-morpholinophenyl)-4-(4-(3-nitrobenzyl)piperazin-1-yl)phthalazin-1-amine (8h)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-nitrobenzyl)piperazine (38.7mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 65%; R_f = 0.42 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H, NH), 8.10 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 6.9 Hz, 1H), 7.96 (d, J = 6.4 Hz, 1H), 7.77 – 7.69 (m, 3H, Ar-H), 7.55–7.45 (m, 3H, Ar-H), 6.88 (d, J = 6.6 Hz, 2H), 3.84 (brs, 4H), 3.69 (s, 2H), 3.36 (brs, 4H), 3.08 (brs, 4H), 2.71 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 148.31, 147.29, 140.52, 135.11, 131.08, 131.01, 129.23, 125.01, 123.78, 123.50, 122.26, 122.03, 116.78, 66.94, 62.16, 53.17, 51.18, 50.97, 50.11; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₁N₇O₃ 526.26, found 526.41.

Synthesis of 3-((4-(4-((4-morpholinophenyl)amino)phthalazin-1-yl)piperazin-1-yl)methyl)benzonitrile (8i)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 3-(piperazin-1-ylmethyl)benzonitrile (35.1mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 83%; R_f = 0.14 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.06–7.92 (m, 2H, Ar-H), 7.82 – 7.71 (m, 2H, Ar-H), 7.66 (s, 1H, NH), 7.61 (d, J = 7.9 Hz, 1H), 7.56–7.47 (m, 3H, Ar-H), 7.44–7.39 (m, 1H, Ar-H), 6.87 (d, J = 10.4 Hz, 2H), 3.92 – 3.76 (m, 4H), 3.62 (s, 2H), 3.45–3.25 (m, 4H), 3.16 – 3.03 (m, 4H), 2.78–2.60 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.48, 149.88, 147.36, 142.55, 139.82, 133.43, 132.45, 131.09, 131.05, 130.95, 130.88, 130.12, 129.11, 125.02, 123.53, 122.10, 118.91, 116.79, 112.34, 66.93, 62.17, 53.15, 51.16, 50.09. LCMS (ESI): Calculated for [M+1]⁺ C₃₀H₃₁N₇O 506.26, found 506.41.

Synthesis of methyl 3-((4-(4-((4-morpholinophenyl)amino)phthalazin-1-yl)piperazin-1-yl)methyl)benzoate (8j)

4- chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and methyl 3-(piperazin-1-ylmethyl)benzoate (41.0mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 91%; R_f = 0.18 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.04–7.99 (m, 2H, Ar-H), 7.98–7.94 (m, 1H, Ar-H), 7.95 – 7.91 (m, 1H, Ar-H), 7.78 – 7.71 (m, 2H, Ar-H), 7.61–7.57 (m, 1H, Ar-H), 7.53–7.46 (m, 2H, Ar-H), 7.40 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 8.0 Hz, 2H), 3.90 (d, J = 4.1 Hz, 3H), 3.86 – 3.82 (m, 4H), 3.66 (s, 2H), 3.43–3.40 (d, J = 9.6 Hz, 4H), 3.11 – 3.07 (m, 4H), 2.75–2.64 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 167.15, 149.81, 147.35, 138.40, 133.77, 131.33, 131.06, 131.02, 130.25, 130.14, 128.45, 128.40, 127.89, 125.11, 123.58, 122.07, 116.82, 66.94, 62.67, 53.11, 52.10, 51.18, 50.11; IR Absorption ν_max/cm⁻¹ 3189.21 (NH), 2840.41 (Ar-H), 1713.53 (C=O), 1287.13 (C-N aromatic), 1234.73 (C-N aliphatic), 1198.11 (C-O ester), 1121.27 (C-O ether); LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₄N₆O₃ 539.21, found 539.27.

Synthesis of 4-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8k)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-methoxybenzyl)piperazine (36.1mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Pale yellow solid; Yield: 36%; R_f = 0.46 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.04 – 7.98 (m, 2H, Ar-H), 7.97 – 7.91 (m, 2H, Ar-H), 7.50 (d, J = 8.1 Hz, 2H), 7.26 – 7.20 (m, 1H, Ar-H), 6.94 (d, J = 7.5 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.82 – 6.77 (m, 1H), 3.85 (t, J = 4.0 Hz, 4H), 3.80 (s, 3H), 3.60 (s, 2H), 3.36 (brs, 4H), 3.08 (t, J = 4.0 Hz, 4H), 2.70 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 159.63, 147.24, 139.60, 131.00, 130.93, 129.17, 125.11, 123.56, 121.99, 121.51, 116.80, 114.51, 112.65, 66.95, 63.04, 55.20, 53.15, 51.24, 50.14; LCMS (ESI): Calculated for [M+1]⁺ C₃₀H₃₄N₆O₂ 511.28, found 511.38.

Synthesis of N-(4-morpholinophenyl)-4-(4-(3-(trifluoromethoxy)benzyl)piperazin-1-yl)phthalazin-1-amine (8l)

4-chloro- N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethoxy)benzyl)piperazine (45.5mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Dark Yellow solid; Yield: 65%; R_f = 0.42 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.03 – 7.97 (m, 2H, Ar-H), 7.74 (p, J = 6.9 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.88 – 3.80 (m, 4H), 3.60 (s, 2H), 3.36 (brs, 4H), 3.13 – 3.04 (m, 4H), 2.69 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 148.30, 148.28, 147.38, 136.70, 131.10, 131.07, 130.34, 125.06, 123.56, 122.14, 120.76, 116.79, 66.94, 62.21, 53.10, 51.16, 50.09; IR Absorption ν_max/cm⁻¹ 3320.08 (NH), 2817.04 (Ar-H), 1254.94 (C-N aromatic), 1220.67 (C-O, alkyl aryl ether), 1117.51 (C-O aliphatic ether), 1157.38 (C-N aliphatic); LCMS (ESI): Calculated for [M+1]⁺ C₃₀H₃₁F₃N₆O₂ 565.25, found 565.45.

Synthesis of 4-(4-(3-methylbenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8m)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-methylbenzyl)piperazine (33.3mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 48%; R_f = 0.46 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.98 (m, 1H, Ar-H), 7.94 (brs, 1H, NH), 7.78–7.68 (m, 2H, Ar-H), 7.49 (d, J = 7.7 Hz, 2H), 7.23 – 7.14 (m, 3H, Ar-H), 7.07 (d, J = 7.3 Hz, 1H), 6.89 (d, J = 9.0 Hz, 2H), 3.85 (t, J = 4.7 Hz, 4H), 3.58 (s, 2H), 3.36 (brs, 4H), 3.09 (t, J = 4.7 Hz, 4H), 2.70 (brs, 4H), 2.34 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 147.24, 137.85, 137.71, 130.99, 130.92, 130.02, 128.11, 127.87, 126.37, 125.13, 121.98, 116.82, 66.95, 63.17, 53.18, 51.20, 50.15, 21.40; LCMS (ESI): Calculated for [M+1]⁺ C₃₀H₃₄N₆O 495.28, found 495.44.

Synthesis of 4-(4-(3,4-dichlorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8n)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3,4-dichlorobenzyl)piperazine (42.9mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 65%; R_f = 0.42 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.14–8.08 (m, 1H, Ar-H), 8.03 – 7.98 (m, 1H, Ar-H), 7.81 – 7.73 (m, 2H, Ar-H), 7.56 – 7.43 (m, 3H, Ar-H), 7.39 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 3.90 – 3.79 (m, 4H), 3.56 (s, 2H), 3.36 (brs, 4H), 3.16 – 3.04 (m, 4H), 2.69 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.74, 138.30, 132.33, 131.46, 131.31, 131.06, 130.87, 130.26, 128.37, 125.10, 123.69, 122.72, 122.70, 122.51, 116.80, 66.92, 61.78, 52.99, 51.08, 49.97; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₀Cl₂N₆O 549.20, found 549.73.

Synthesis of 4-(4-(3,4-difluorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8o)

4-chloro-N-(4- morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3,4-difluorobenzyl)piperazine (37.1mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Off-white solid; Yield: 53%; R_f = 0.36 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.02 – 7.94 (m, 2H, Ar-H), 7.78 – 7.72 (m, 2H, Ar-H), 7.50 (d, J = 8.5 Hz, 2H), 7.21 (dd, J = 9.6, 8.1 Hz, 1H), 7.11 – 7.04 (m, 2H, Ar-H), 6.90 (d, J = 9.0 Hz, 2H), 3.87 – 3.83 (m, 4H), 3.55 (s, 2H), 3.35 (brs, 4H), 3.12 – 3.07 (m, 4H), 2.68 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 149.03, 148.97, 148.17, 147.38, 135.24, 135.21, 135.17, 135.15, 131.09, 131.04, 125.07, 124.72, 124.67, 124.65, 123.56, 122.08, 117.68, 116.84, 66.95 (2C), 61.96 (2C), 53.06 (2C), 51.18 (2C), 50.11; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₀F₂N₆O 517.25, found 517.36

Synthesis of 4-(4-(2,6-dichlorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8p)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(2,6-dichlorobenzyl)piperazine (42.9mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 69%; R_f = 0.61 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.37 (p, J = 8Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 6.75 (t, J = 8.0 Hz, 1H), 6.51 (d, J = 8.7 Hz, 2H), 3.50–3.44 (m, 6H), 3.00–2.85 (brm, 4H), 2.71 (t, J = 4 Hz, 4H), 2.44 (t, J = 4 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 147.40, 137.02, 134.19, 131.11, 131.06, 128.83, 128.35, 125.22, 123.67, 122.13, 116.85, 66.95, 56.50, 53.01, 51.24, 50.10; LCMS (ESI): Calculated for [M]⁺ C₂₉H₃₀Cl₂N₆O 549.20, found 549.60.

Synthesis of 4-(4-(2,4-difluorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8q)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(2,4-difluorobenzyl)piperazine (37.1mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. White solid; Yield: 31%; R_f = 0.36 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H, NH), 8.02 – 7.98 (m, 1H, Ar-H), 7.84 – 7.79 (m, 2H, Ar-H), 7.47 (d, J = 8.8 Hz, 3H), 6.93 (d, J = 9.0 Hz, 5H), 3.86 (t, J = 8.8 Hz, 4H), 3.73 (s, 2H), 3.42 (brs, 4H), 3.13 (t, J = 4.0 Hz, 4H), 2.80 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 167.20, 160.15, 149.97, 148.47, 132.25, 131.75, 128.06, 124.26, 116.82, 113.41, 103.67, 66.87 (2C), 54.39 (2C), 52.20 (2C), 50.58 (2C), 49.73; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₀F₂N₆O 517.25, found 517.39.

Synthesis of 4-(4-(3-chloro-4-fluorobenzyl)piperazin-1-yl)-N-(4-morpholinophenyl)phthalazin-1-amine (8r)

4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (50 mg, 0.146mmol) was dissolved in n-butanol (1mL) and 1-(3-chloro-4-fluorobenzyl)piperazine (40mg, 0.175mmol) ) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Light Yellow solid; Yield: 77%; R_f = 0.40 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 7.97 (d, J = 17.9 Hz, 2H), 7.80 – 7.70 (m, 2H, Ar-H), 7.50 (d, J = 8.5 Hz, 2H), 7.41 (dd, J = 7.2, 2.1 Hz, 1H), 7.24 – 7.19 (m, 1H, Ar-H), 7.08 (t, J = 8.7 Hz, 1H), 6.89 (d, J = 8.9 Hz, 2H), 3.89 – 3.79 (m, 4H), 3.54 (brs, 2H), 3.35 (s, 4H), 3.14 – 3.05 (m, 4H), 2.67 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.44, 155.97, 147.33, 135.22, 135.18, 131.07, 131.01, 130.98, 128.64, 128.57, 125.06, 123.54, 122.04, 120.76, 120.59, 116.81, 116.38, 116.17, 77.32, 77.21, 77.00, 76.68, 66.95, 61.83, 53.07, 51.18, 50.12, 15.26; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₃₀ClFN₆O 533.22, found 533.47.

Synthesis of N-(2-methoxyethyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine(10a)

4-chloro-N-(2-methoxyethyl)phthalazin-1-amine (9a) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Dark yellow solid; Yield: 49%; R_f = 0.33 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.01 (t, J = 4.1 Hz, 1H), 7.82 (brs, 1H, NH), 7.77 – 7.69 (m, 2H, Ar-H), 7.62 (s, 1H), 7.58 (d, J = 6.7 Hz, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.44 (t, J = 7.3 Hz, 1H), 3.85 (s, 2H), 3.71 (t, J = 4.9 Hz, 2H), 3.66 (s, 2H), 3.40 (s, 3H), 3.36 (s, 4H), 2.70 (s, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 160.70, 155.40, 151.78, 139.13, 138.75, 132.39, 132.17, 131.26, 130.75, 130.43, 128.81, 128.73, 125.71, 125.67, 125.53, 125.51, 125.47, 124.99, 124.22, 124.18, 124.02, 123.98, 123.36, 122.82, 121.13, 62.52, 58.86, 53.11, 51.18, 45.57, 39.89.; LCMS (ESI): Calculated for [M+1]⁺ C₂₃H₂₆F₃N₅O 446.21, found 446.26.

Synthesis of N¹,N¹-dimethyl-N²-(4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-yl)ethane-1,2-diamine(10b)

N¹-(4-chlorophthalazin-1-yl)-N²,N²-dimethylethane-1,2-diamine (9b) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 42%; R_f = 0.07 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.12–8.04 (m, 1H, Ar-H), 8.04–7.95 (m, 1H, Ar-H), 7.80 – 7.70 (m, 2H, Ar-H), 7.62 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.80 (brs, 2H), 3.66 (s, 2H), 3.35 (brs, 4H), 2.85 (s, 2H), 2.70 (brs, 4H), 2.44 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 155.46, 152.07, 139.28, 132.37, 131.09, 130.57, 128.70, 125.68, 124.71, 123.95, 123.26, 122.55, 121.09, 62.57, 58.23, 53.22 (2C), 51.25 (2C), 44.80 (2C), 38.67 (2C), 29.68; LCMS (ESI): Calculated for [M+1]⁺ C₂₄H₂₉F₃N₆ 459.24, found 459.14.

Synthesis of 1-(4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-yl)piperidin-4-ol (10c)

1-(4-chlorophthalazin-1-yl)piperidin-4-ol (9c) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Orange solid; Yield: 65%; R_f = 0.12 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.02 – 7.96 (m, 2H, Ar-H), 7.77 – 7.70 (m, 2H, Ar-H), 7.62 (s, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.97–3.90 (m, 1H), 3.75–3.67 (m, 2H), 3.46 (brs, 4H), 3.15 (t, J = 10.4 Hz, 2H), 2.72 (brs, 4H), 2.15–2.05 (m, 2H), 1.90–1.66 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.34, 157.82, 139.02, 132.46, 130.79, 130.59, 128.76, 125.75, 125.73, 125.09, 124.93, 124.16, 124.05, 123.97, 123.74, 68.24 (2C), 62.53 (2C), 53.05 (2C), 50.91, 49.05 (2C), 34.68 (2C); LCMS (ESI): Calculated for [M+1]⁺ C₂₅H₂₈F₃N₅O 472.23, found 472.39.

Synthesis of N-(1-methylpiperidin-4-yl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10d)

4- chloro-N-(1-methylpiperidin-4-yl)phthalazin-1-amine (9d) (50 mg, 0.10mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (29.3mg, 0.12mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 82%; R_f = 0.03 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.02 – 7.95 (m, 1H, Ar-H), 7.78–7.65 (m, 3H, Ar-H), 7.59 (s, 1H, NH), 7.55 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 4.27 (brs, 1H), 3.63 (s, 2H), 3.32 (brs, 4H), 2.95–2.91 (m, 2H), 2.67 (brs, 4H), 2.33 (s, 3H), 2.24 (t, J = 3 Hz, 4H), 1.68 (dd, J = 22.0, 10.5 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 155.30, 151.12, 139.28, 132.37, 132.36, 130.68, 130.66, 130.35, 128.68, 125.65, 125.54, 124.89, 124.18, 123.91, 123.17, 122.83, 121.10, 120.63, 62.54, 54.70, 53.24, 51.28, 47.46, 45.84, 32.00; LCMS (ESI): Calculated for [M+1]⁺ C₂₆H₃₁F₃N₆ 485.26, found 485.36.

Synthesis of N-cyclohexyl-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10e)

4-chloro-N-cyclohexylphthalazin-1-amine (9e) (50 mg, 0.106mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (31.2mg, 0.127mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Dark yellow solid; Yield: 64%; R_f = 0.82 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.01–7.90 (m, 2H, Ar-H), 7.77 – 7.67 (m, 2H, Ar-H), 7.60 (s, 1H, NH), 7.56 (d, J = 7.5 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 5.39 (brs, 1H), 4.26 (s, 1H), 3.64 (s, 2H), 3.32 (s, 4H), 2.68 (s, 4H), 2.19 (d, J = 10.8 Hz, 2H), 1.73 (d, J = 12.4 Hz, 2H), 1.62 (d, J = 13.0 Hz, 1H), 1.45 – 1.29 (m, 4H), 1.24 – 1.16 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 154.83, 150.82, 139.10, 132.42, 131.03, 130.87, 130.54, 128.72, 125.61, 125.43, 124.92, 124.18,123.97, 123.37, 121.88, 120.93, 62.50 (2C), 53.14(2C), 51.16 (2C), 50.23 (2C), 33.22 (2C), 25.11 (2C); LCMS (ESI): Calculated for [M+1]⁺ C₂₆H₃₀F₃N₅ 470.25, found 470.35.

Synthesis of 1-(piperidin-1-yl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazine (10f)

1-chloro-4-(piperidin-1-yl)phthalazine (9f) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 71%; R_f = 0.85 (50% EtOAc/Hexane on alumina);; ¹H NMR (400 MHz, CDCl₃) δ 8.03 – 7.96 (m, 2H, Ar-H), 7.75 – 7.69 (m, 2H, Ar-H), 7.62 (s, 1H, NH), 7.59 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.66 (s, 2H), 3.45 (brs, 4H), 3.37 – 3.28 (m, 4H), 2.72 (brs, 4H), 1.85–1.75 (m, 4H), 1.72–1.64 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 159.00, 157.59, 132.48, 131.28, 130.61, 130.60, 130.58, 128.77, 125.77, 125.30, 124.81, 124.11, 124.16, 124.07, 123.75, 62.52 (2C), 53.04 (2C), 52.47 (2C), 50.86 (2C), 26.14, 24.66; LCMS (ESI): Calculated for [M+1]⁺ C₂₅H₂₈F₃N₅ 456.23, found 456.36.

Synthesis of 1-(2-((4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-yl)amino)ethyl)piperidin-4-ol (10g)

1-(2- ((4-chlorophthalazin-1-yl)amino)ethyl)piperidin-4-ol (9g) (50 mg, 0.097mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (28.4mg, 0.116mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 53%; R_f = 0.03 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, DMSO-d6) δ 8.40 – 8.34 (m, 1H, Ar-H), 8.08 – 8.02 (m, 1H, Ar-H), 8.01 – 7.96 (m, 2H, Ar-H), 7.89 – 7.81 (m, 1H, Ar-H), 7.78 (brs, 1H), 7.68 (d, J = 8.0Hz 1H), 7.65 – 7.56 (m, 1H, Ar-H), 3.68 (s, 4H), 3.42 (s, 4H), 3.17 (s, 2H), 2.90 (s, 2H), 2.74 (brs, 2H), 2.66 (brs, 2H), 2.35 – 2.27 (m, 1H), 1.76 (d, J = 11.4 Hz, 3H), 1.57 – 1.36 (m, 3H); ¹³C NMR (101 MHz, DMSO-d6) δ 154.48, 152.41, 144.50, 140.17, 133.36, 132.98, 129.73, 125.67, 125.63, 124.91, 124.58, 124.21, 123.33, 120.64, 120.43, 66.01 (2C), 61.76 (2C), 56.68(2C), 56.32 (2C), 53.11, 51.54, 51.23, 34.10; LCMS (ESI): Calculated for [M+1]⁺ C₂₇H₃₃F₃N₆O 515.27, found 515.20.

Synthesis of 4-(2-((4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-yl)amino)ethyl)thiomorpholine 1,1- dioxide (10h)

4-(2-((4-chlorophthalazin-1-yl)amino)ethyl)thiomorpholine 1,1-dioxide (9h) (50 mg, 0.091mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (26.6mg, 0.109mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Golden solid; Yield: 62%; R_f = 0.06 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05 – 8.01 (m, 1H, Ar-H), 7.81 – 7.73 (m, 3H, Ar-H), 7.61 (s, 1H, NH), 7.57 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.75 (dd, J = 11.0, 5.5 Hz, 2H), 3.66 (s, 2H), 3.41 – 3.28 (m, 4H), 3.09 (dd, J = 15.7, 6.5 Hz, 8H), 2.91 (t, J = 5.9 Hz, 2H), 2.70 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.68, 151.72, 139.22, 132.37, 131.01, 130.56, 128.72, 125.67, 125.54, 124.19, 123.97, 123.18, 121.08, 120.75, 120.13, 62.55 (2C), 55.12 (2C), 53.19 (2C), 51.27 (2C), 50.66, 43.27, 38.73; LCMS (ESI): Calculated for [M+1]⁺ C₂₆H₃₁F₃N₆O₂S 549.22, found 549.31.

Synthesis of N-(2-(piperidin-1-yl)ethyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10i)

4- chloro-N-(2-(piperidin-1-yl)ethyl)phthalazin-1-amine (9i) (50 mg, 0.10mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (29.4mg, 0.12mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Dark yellow solid; Yield: 51%; R_f = 0.08 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.02 – 7.93 (m, 2H, Ar-H), 7.78 – 7.69 (m, 2H, Ar-H), 7.61 (s, 1H), 7.57 (d, J = 7.3 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 3.73 (t, J = 8.0 Hz, 2H), 3.65 (s, 2H), 3.34–3.33 (m, 4H), 2.77 (t, J = 6.0 Hz, 2H), 2.69 (brs, 3H), 2.55 (brs, 4H), 1.70 – 1.58 (m, 4H), 1.48 (brs, 2H), 1.23 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 155.25, 152.25, 139.26, 132.39, 130.90, 130.83, 130.54, 128.70, 125.69, 124.71, 124.19, 123.93, 123.21, 122.01, 121.08, 62.58 (2C), 57.14 (2C), 54.13 (2C), 53.24 (2C), 51.23, 38.00, 25.58, 24.02; LCMS (ESI): Calculated for [M+1]⁺ C₂₇H₃₃F₃N₆ 499.28, found 499.23.

Synthesis of N-(1H-pyrazol-5-yl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10j)

4-chloro-N- (1H-pyrazol-5-yl)phthalazin-1-amine (9j) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Bright yellow solid; Yield: 54%; R_f = 0.55 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J = 6.9 Hz, 1H), 7.88 – 7.81 (m, 1H, Ar-H), 7.74 – 7.64 (m, 2H, Ar-H), 7.62 (brs, 1H, NH), 7.57 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 6.38 (s, 1H), 3.66 (s, 2H), 3.25 (brs, 4H), 2.68 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 146.61, 139.14, 132.34, 131.36, 131.29, 130.64, 130.48, 129.35, 128.74, 125.65, 125.53, 124.47, 124.37, 124.32, 124.08, 124.02, 123.97, 122.82, 62.50 (2C), 53.03 (2C), 50.88; LCMS (ESI): Calculated for [M+1]⁺ C₂₃H₂₂F₃N₇ 454.19, found 454.32.

Synthesis of 4-((4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-yl)amino)phenol (10k)

4-((4-chlorophthalazin-1-yl)amino)phenol (9k) (50 mg, 0.11mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (32.9mg, 0.13mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 76%; R_f = 0.08 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.04 – 7.96 (m, 1H, Ar-H), 7.78–7.73 (m, 1H, Ar-H), 7.56 (s, 2H), 7.48 (d, J = 7.9 Hz, 4H), 7.41 (dd, J = 14.5, 6.8 Hz, 3H), 6.88 (d, J = 8.4 Hz, 1H), 3.51 (s, 2H), 2.96 – 2.83 (m, 4H), 2.44 (brs, 4H); ¹³C NMR (101 MHz, DMSO) δ 155.45, 152.96, 151.17, 140.18, 133.48, 132.99, 131.77, 131.49, 129.75, 129.41, 126.11, 125.65, 124.63, 124.22, 123.71, 123.40, 123.14, 122.80, 120.91, 115.20, 61.76 (2C), 53.09 (2C), 51.52; LCMS (ESI): Calculated for [M+1]⁺ C₂₆H₂₄F₃N₅O 480.20, found 480.38.

Synthesis of N-(4-methoxyphenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10l)

4-chloro-N-(4-methoxyphenyl)phthalazin-1-amine (9l) (50 mg, 0.10mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (29.4mg, 0.12mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 69%; R_f = 0.82 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 7.3 Hz, 1H), 7.96 (d, J = 6.7 Hz, 1H), 7.78 – 7.70 (m, 2H, Ar-H), 7.61 (s, 1H, NH), 7.57 (d, J = 7.4 Hz, 1H), 7.54–7.40 (m, 4H, Ar-H), 6.86 (d, J = 7.1 Hz, 2H), 3.77 (s, 3H), 3.65 (s,2H), 3.36 (brs, 4H), 2.70 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.52, 149.88, 139.22, 132.37, 131.06, 131.00, 130.40, 128.72, 125.66, 125.03, 124.42, 123.97, 123.95, 123.91, 123.54, 122.47, 122.02, 114.30, 62.53, 55.52 (2C), 53.16 (2C), 51.20; LCMS (ESI): Calculated for [M+1]⁺ C₂₇H₂₆F₃N₅O 494.21, found 494.38.

Synthesis of N-(4-(piperidin-1-yl)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10m)

4-chloro-N-(4-(piperidin-1-yl)phenyl)phthalazin-1-amine (9m) (50 mg, 0.091mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (26.8mg, 0.109mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated and reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 44%; R_f = 0.84 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.03 – 7.99 (m, 1H, Ar-H), 7.98–7.90 (m, 1H, Ar-H), 7.77 – 7.70 (m, 2H, Ar-H), 7.62 (s, 1H, NH), 7.58 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.47–7.38 (m, 3H, Ar-H), 6.93 (d, J = 9.0 Hz, 2H), 3.66 (s, 2H), 3.36 (brs, 4H), 3.14 – 3.03 (m, 4H), 2.70 (brs, 4H), 1.70 (dt, J = 11.2, 5.7 Hz, 4H), 1.57 – 1.51 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 160.70, 148.56, 139.21, 132.38, 132.37, 131.03, 130.99, 130.57 (q, J = 32 Hz), 128.72, 125.67 (q, J = 4 Hz), 125.04, 124.19 (q, J = 271 Hz), 123.97 (q, J = 4 Hz), 123.58, 122.03, 117.68, 113.74, 62.54, 53.17, 51.49, 51.19, 25.92, 24.22; LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₃F₃N₆ 547.28, found 547.47.

Synthesis of N-(4-(4-methylpiperazin-1-yl)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10n)

4-chloro-N-(4-(4-methylpiperazin-1-yl)phenyl)phthalazin-1-amine (9n) (50 mg, 0.089mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (26.08mg, 0.106mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated, and reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 80%; R_f = 0.196 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.90 (m, 2H, Ar-H), 7.77 – 7.69 (m, 2H, Ar-H), 7.61 (s, 1H, NH), 7.56 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.47 – 7.40 (m, 2H, Ar-H), 6.90 (d, J = 9.0 Hz, 2H), 3.64 (s, 2H), 3.35 (s, 4H), 3.16 – 3.10 (m, 4H), 2.69 (s, 4H), 2.60 – 2.54 (m, 4H), 2.33 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 150.04, 147.19, 139.22, 132.37, 131.01, 130.92, 130.55 (q, J = 64 Hz), 128.71, 128.58, 125.65 (q, J = 4 Hz), 124.97, 124.19 (q, J = 271 Hz), 123.95 (q, J = 4 Hz), 123.50, 121.99, 121.85, 117.14, 62.53, 55.06, 53.17, 51.21, 49.77, 46.01; IR Absorption ν_max/cm⁻¹ 3303.42 (NH), 2922.50, 2808.82 (Ar-H), 1325.60 (C-N, aromatic), 1159.61 (C-F), 1091.85 (C-N aliphatic); LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₄F₃N₇ 562.29, found 562.44.

Synthesis of N-(4-(4-ethylpiperazin-1-yl)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10o)

4-chloro-N-(4-(4-ethylpiperazin-1-yl)phenyl)phthalazin-1-amine (9o) (50 mg, 0.087mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5mg, 0.104mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated, and reaction was purified by Flash chromatography on alumina. Dark green solid; Yield: 56%; R_f = 0.28 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.97 (m, 1H, Ar-H), 7.76 – 7.72 (m, 2H, Ar-H), 7.62 (s, 1H, NH), 7.57 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 8.0 Hz, 3H),7.46–7.34 (m, 2H), 6.93 (d, J = 9.0 Hz, 2H), 3.66 (s, 2H), 3.36 (brs, 4H), 3.23 – 3.15 (m, 5H), 2.75–2.65 (m, 8H), 2.52 (q, J = 8.0 Hz, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.21, 139.24, 132.35, 131.02, 130.93, 130.73, 130.57 (q, J = 32 Hz), 128.71, 125.67 (q, J = 4 Hz), 125.07, 124.18 (q, J = 271 Hz), 123.97 (q, J = 4 Hz), 121.96, 118.74, 117.30, 116.15, 62.55, 53.18, 52.70, 52.34, 51.21, 49.70, 11.71; IR Absorption ν_max/cm⁻¹ 3309.53 (NH), 2920.83, 2850.03 (Ar-H), 1326.05 (C-N aromatic), 1118.73 (C-N aliphatic), 1071.14 (C-F); LCMS (ESI): Calculated for [M+1]⁺ C₃₂H₃₆F₃N₇ 576.30, found 576.41.

Synthesis of N-(4-(3-methylmorpholino)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10p)

4-chloro N-(4-(3-methylmorpholino)phenyl)phthalazin-1-amine (9p) (50 mg, 0.087mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5mg, 0.104mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated, and reaction was purified by Flash chromatography on alumina. Dark green solid; Yield: 51%; R_f = 0.54 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.09 (brs, 1H, NH), 8.02–7.99 (m, 1H, Ar-H), 7.77 – 7.74 (m, 2H, Ar-H), 7.61 (s, 1H), 7.58 (d, J = 7.3 Hz, 1H), 7.53–7.50 (m, 1H, Ar-H), 7.47–7.38 (m, 3H, Ar-H), 6.75 (d, J = 8.8 Hz, 2H), 3.66 (s, 2H), 3.35 (m, 4H), 3.33 (s, 2H), 2.91 (m, 4H), 2.70 (m, 4H), 1.23 (s, 3H), 1.01 (d, J = 6.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 149.82, 149.79, 147.30, 139.07, 132.38, 131.25, 131.18, 130.60, 128.74, 125.69, 125.11, 124.02, 123.96, 123.66, 122.31, 116.87, 71.65 (2C), 62.51 (2C), 55.54 (2C), 53.09, 51.14, 19.07; IR Absorption ν_max/cm⁻¹ 3311.32 (NH), 2814.57 (Ar-H), 1325.67 ( C-N aromatic), 1108.56 (C-N aliphatic), 1070.77 ( C-O aliphatic ether); LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₃F₃N₆O 563.27, found 563.36.

Synthesis of N-(4-(2-methylmorpholino)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10q)

4-chloro-N-(4-(2-methylmorpholino)phenyl) phthalazin-1-amine (9q) (50 mg, 0.087mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5mg, 0.104mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated, and reaction was purified by Flash chromatography on alumina. Dark brown solid; Yield: 76%; R_f = 0.55 (50% EtOAc/Hexane on alumina);¹H NMR (400 MHz, CDCl₃) δ 8.06 – 7.98 (m, 2H, Ar-H), 7.79 – 7.73 (m, 2H, Ar-H), 7.62 (s, 1H, NH), 7.60–7.56 (m, 1H, Ar-H), 7.53 – 7.43 (m, 4H, Ar-H), 6.90 (d, J = 8.9 Hz, 2H), 3.81 (m, 2H), 3.66 (s, 2H), 3.42 – 3.32 (m, 6H), 2.71 (m, 4H), 2.41 – 2.35 (m, 2H), 1.24 (d, J = 6.3 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 149.82, 149.79, 147.30, 139.07, 132.39, 132.38, 131.25, 131.18, 130.6, 128.74, 125.69, 125.11, 124.17, 124.02, 123.66, 122.31, 116.87, 71.65, 62.51 (2C), 55.54 (2C), 53.09 (2C), 51.14, 19.07 (2C). LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₃F₃N₆O 563.27, found 563.36.

Synthesis of N-(4-(2,6-dimethylmorpholino)phenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)phthalazin-1-amine (10r)

4-chloro- N-(4-(2,6-dimethylmorpholino)phenyl) phthalazin-1-amine (9r) (50 mg, 0.087mmol) was dissolved in n-butanol (1mL) and 1-(3-(trifluoromethyl)benzyl)piperazine (25.5mg, 0.104mmol) was added and was heated overnight at 150°C. Next, n-butanol was concentrated, and reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 56%; R_f = 0.41 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.05–8.00 (m, 2H, Ar-H), 7.79 – 7.74 (m, 2H, Ar-H), 7.62 (s, 1H, NH), 7.59 – 7.57 (m, 1H, Ar-H), 7.53–7.48 (m, 3H, Ar-H), 7.45 – 7.40 (m, 1H, Ar-H), 6.90 (d, J = 8.0 Hz, 2H), 4.03 – 3.97 (m, 1H), 3.83 – 3.75 (m, 2H), 3.66 (s, 2H), 3.56 (s, 1H), 3.38 – 3.35 (m, 4H), 2.81 – 2.75 (m, 1H), 2.74 – 2.63 (m, 4H), 2.46 – 2.40 (m, 2H), 1.23 (d, J = 8.0 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 160.69, 147.43, 139.06, 138.75, 132.39, 132.16, 131.24, 131.18, 130.6, 128.80, 128.74, 125.69, 125.34, 125.11, 124.20, 124.17, 124.02, 123.64, 122.28, 116.87, 71.77, 62.51 (2C), 55.54 (2C), 53.09 (2C), 51.14, 19.05 (2C); IR Absorption ν_max/cm⁻¹ 3329.72 (NH), 2813.99 (Ar-H), 1325.36 (C-N aromatic), 1235.23 ( C-N aliphatic), 1071.22 (C-O, aliphatic ether); LCMS (ESI): Calculated for [M+1]⁺ C₃₂H₃₅F₃N₆O 577.29, found 577.31.

Synthesis of N-(4-morpholinophenyl)-4-(4-((3-(trifluoromethyl)benzyl)amino)phenyl)phthalazin-1-amine (11)

In 5mL oven dried microwave vial 4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (150mg, 0.44mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-(3-(trifluoromethyl)benzyl)aniline (249mg, 0.66mmol) was charged in DMF:H₂O (4:1) 2 mL and added Cesium carbonate (430.4mg, 1.32 mmol). The reaction mixture was vigorously degassed with nitrogen for ten minutes. Following, 3.0 mol% of Pd(PPh₃)4 was added and the reaction was irradiated (Biotage 400 MW) at 115°C for 1h. The reaction mixture was filtered through a pad of Celite with CH₂Cl₂ washing. The combined filtrate was concentrated and the residue was washed with water and CH₂Cl₂ followed by Flash chromatography on alumina. Brown solid; Yield: 29%; R_f = 0.42 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.75 – 7.67 (m, 2H, Ar-H), 7.63 (s, 1H, NH), 7.75–7.44 (m, 7H, Ar-H), 6.88 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 8.6 Hz, 2H), 4.43 (s, 2H), 3.83 (t, J = 4Hz, 4H), 3.07 (t, J = 4Hz, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 148.17, 147.42, 140.34, 131.29, 131.12, 130.93, 130.77, 129.12, 127.13, 126.83, 125.76, 124.09, 124.10, 123.99, 122.23, 116.79, 112.59, 66.93 (2C), 50.01 (2C), 47.66; LCMS (ESI): Calculated for [M+1]⁺ C₃₂H₂₈F₃N₅O 556.23, found 556.46.

Synthesis of N-(4-morpholinophenyl)-4-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)phthalazin-1-amine (12)

In 5mL oven dried microwave vial 4-chloro-N-(4-morpholinophenyl)phthalazin-1-amine (7) (150mg, 0.44mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(3-(trifluoromethyl)benzyl)-1H-pyrazole (232.2mg, 0.66mmol) was charged in DMF:H₂O (4:1) 2 mL and added Cesium carbonate (430.4mg, 1.32 mmol). The reaction mixture was vigorously degassed with nitrogen for ten minutes. Following, 3.0 mol% of Pd(PPh₃)4 was added and the reaction was irradiated (Biotage 400 MW) at 115°C for 1h. The reaction mixture was filtered through a pad of Celite with CH₂Cl₂ washing. The combined filtrate was concentrated and the residue was washed with water and CH₂Cl₂ followed by Flash chromatography on alumina. Orange solid; Yield: 65%; R_f = 0.28 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.19 – 8.14 (m, 1H, Ar-H), 8.09 – 8.04 (m, 1H, Ar-H), 8.00 (s, 1H, NH), 7.95 (s, 1H), 7.84 −7.76 (m, 2H, Ar-H), 7.56 (t, J = 6.2 Hz, 4H), 7.46 (d, J = 5.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 5.43 (s, 2H), 3.86 – 3.82 (m, 4H), 3.11 – 3.08 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 147.63, 139.98, 137.05, 131.83, 131.45, 131.42, 131.40, 131.14, 131.13, 131.08, 130.76, 130.00, 129.48, 126.48, 125.96, 125.13, 124.54, 122.31, 121.76, 120.36, 119.28, 116.68, 66.91 (2C), 55.73 (2C), 49.95; LCMS (ESI): Calculated for [M+1]⁺ C₂₉H₂₅F₃N₆O 531.21, found 531.35.

Synthesis of N-(4-morpholinophenyl)-1-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)isoquinolin-4-amine (16)

In 5mL oven dried microwave vial 4-bromo-1-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)isoquinoline (15) (100mg, 0.22mmol) and 4-morpholinoaniline (59.3mg, 0.33mmol) was charged in 2mL dioxane and added potassium tert-butoxide (74.8mg, 0.66mmol) The reaction mixture was vigorously degassed with nitrogen for ten minutes. Following, 0.3% of Pd₂dba₃ and 0.9% (±)-BINAP of was added and the reaction was heated at 110°C for overnight. Next solvent was then removed by rotavap, and the resulting residue dissolved in dichloromethane (30 mL) with rapid stirring. Water (30 mL) was then added to the resulting mixture, and the organic layer was separated. The aqueous layer was then extracted with dichloromethane, and the combined organic layers were dried over Na₂SO₄. Reaction was purified by Flash chromatography on alumina. Yellow solid; Yield: 42%; R_f = 0.25 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.1 Hz, 1H), 8.02 (s, 1H, NH), 7.88 (d, J = 8.2 Hz, 1H), 7.65 (s, 1H, NH), 7.57 (t, J = 7.6 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.44 (t, J = 7.7 Hz, 1H), 6.80 (s, 4H), 3.83 (t, J = 8.0 Hz, 4H), 3.68 (s, 2H), 3.39 (brs, 4H), 3.03 (t, J = 8.0 Hz, 4H), 2.73 (brs, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 157.86, 145.44, 139.40, 139.34, 133.71, 133.40, 132.33, 130.59, 129.86, 129.45, 128.70, 126.24, 125.96, 125.64, 123.95, 122.38, 122.24, 121.50, 117.76, 117.64, 67.01 (2C), 62.50 (2C), 53.32 (2C), 51.44 (2C), 50.61; LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₂F₃N₅O 548.26, found 548.31.

Synthesis of N-(4-morpholinophenyl)-4-(4-(3-(trifluoromethyl)benzyl)piperazin-1-yl)isoquinolin-1-amine (18)

In 5mL oven dried microwave vial 4-bromo-N-(4-morpholinophenyl)isoquinolin-1-amine (17) (100mg, 0.26mmol) and 1-(3-(trifluoromethyl)benzyl)piperazine (95.1mg, 0.39mmol) was charged in 2mL dioxane and added potassium tert-butoxide (87.4mg, 0.78mmol) The reaction mixture was vigorously degassed with nitrogen for ten minutes. Following, 0.3% of Pd₂dba₃ and 0.9% (±)-BINAP of was added and the reaction was heated at 110°C for overnight. Next solvent was then removed by rotavap, and the resulting residue dissolved in dichloromethane (30 mL) with rapid stirring. Water (30 mL) was then added to the resulting mixture, and the organic layer was separated. The aqueous layer was then extracted with dichloromethane, and the combined organic layers were dried over Na₂SO₄. Reaction was purified by Flash chromatography on alumina. Brown solid; Yield: 18%; R_f = 0.84 (50% EtOAc/Hexane on alumina); ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H, NH), 7.67 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 4Hz, 1H), 7.57 (brs, 1H), 7.54 – 7.50 (m, 2H, Ar-H), 7.46 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 5.10 (s, 1H), 3.90 – 3.82 (m, 4H), 3.66 (s, 4H), 3.33 (s, 2H), 3.16 – 3.00 (m, 8H); LCMS (ESI): Calculated for [M+1]⁺ C₃₁H₃₂F₃N₅O 548.26, found 548.40.

Highlights:

• TGFβ is one of few essential druggable targets for designing small-molecule inhibitors to target the tumor microenvironment.

• A series of phthalazine scaffolds were designed and investigated as a small-molecule inhibitor that targets the TGFβ pathway via a non-receptor-kinase mechanism.

• Compound (10p) exhibited promising TGFβ pathway inhibition at IC₅₀ of 0.11 ± 0.02 µM and was confirmed to be non-cytotoxic up to 12 µM, with a selectivity index of approximately 112-fold.

• These pthalazine inhibitors were confirmed to reduce smad phosphorylation induced by the TGFβ pathway via western blot analysis

• Most active compounds were screened against various kinases to establish selectivity for inhibiting the TGFβ pathway via a non-receptor-kinase mechanism.

ABBREVIATIONS

TGFβ

transforming growth factor-beta

TGFβR

Transforming growth factor-beta receptor

SAR

Structure activity relationship

NK

Natural killer

ALK

Activin Receptor-Like Kinase

ATP

Adenosine Triphospate

SMAD

small mothers against decapentaplegic

MW

Microwave

rt

room temperature