Synthesis of Novel Phthalazinedione-Based Derivatives with Promising Cytotoxic, Anti-bacterial, and Molecular Docking Studies as VEGFR2 Inhibitors.

The parent ester methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino] has 18 compounds. The starting material for alkanoates, their corresponding hydrazides, hydrazones, and dipeptides were produced by chemoselective O-alkylation of 2-phenyl-2,3-dihydrophthalazine-1,4-dione with ethyl chloroacetate(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy) acetic acid methyl ester. The starting ester was hydrazinolyzed, then azide coupled with amino acid ester hydrochloride to produce several parent esters, and then hydrazinolyzed to produce parent hydrazides. These hydrazides were used to make a series of dipeptides by reacting them with amino acid ester hydrochloride under azide coupling conditions, and they were also condensed with a number of aldehydes to make the hydrazones. These derivatives were subjected to cytotoxicity against HCT-116 and MDA-MB-231 cells and anti-bacterial and molecular docking studies. Results indicated that the tested compounds, especially 7c and 8b with the phenyl phthalazinone moieties, had promising cytotoxicity against the HCT-116 cells with IC50 values of 1.36 and 2.34 μM, respectively. Additionally, the promising compounds 7c and 8b exhibited poor cytotoxicity against WISH cells with much higher IC50 values, so they were safe against normal cells. Compound 8c exhibited potent anti-bacterial activity with inhibition zones of 12 and 11 mm against Staphylococcus aureus and Escherichia coli, respectively. The molecular docking results of compounds 7c and 8b revealed a good binding disposition and the ligand-receptor interactions like the co-crystallized ligand of the VEGFR2 protein, which may be the proposed mode of action. Finally, compounds 7c and 8b exhibited good ADME pharmacokinetics with good drug-likeness parameters. Hence, detailed studies for the mechanism of action of such compounds are highly recommended for the development of new potent anti-cancer and anti-bacterial agents.

Introduction

Cancer is a leading cause of death and a major determinant of life expectancy worldwide. Liver, lung, and gastric cancer for males and breast, lung, and colorectal cancer for females are the leading causes of cancer death.[1] Our research group has recently directed our attention on finding new anti-cancer drugs,[2,3] where anti-cancer drug research is never-ending, to develop products with a lower toxicity and greater selectivity for tumor cells. Phthalazinedione and its derivatives have been studied by chemists and pharmacologists for their broad-spectrum natural exercises and applications, which include antitumor,[4] cytotoxic,[5] anticonvulsant, cardiotonic, vasorelaxant, antimicrobial, and anti-inflammatory properties.[6]

Several phthalazine precursors have emerged as promising and appealing candidates in the development of novel anti-cancer agents against breast, liver, and colon cancer (Figure A).[7−11] Because VEGFR2 is highly expressed in several solid tumors and plays an important role in the apoptosis process, VEGFR-2 inhibition has emerged as a promising approach for developing new therapies for many apoptosis-dependent cancers. Some promising phthalazine derivatives, such as PTK 787, ZD 6474, and Vandetanib, were investigated as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2) (Figure B).

Figure 1

(A) Some common phthalazine-based compounds are used as anti-cancer drugs. (B) Some common phthalazine-based compounds as VEGFR2 inhibitors. (C) Design strategy for the synthesized compounds.

So, the aim of this study is to design and synthesize novel phthalazinedione derivatives, maintaining the essential pharmacophoric regions of phthalazine, hydrogen bond donors/acceptors, and the lipophilic moieties targeting VEGFR2 inhibition, against breast and colon cancer.

Results and Discussion

Chemistry

Our research team quickly discovered that chemoselective alkylation of amides and thioamides could be regulated.[12−18] We agreed to extend our results to the structure modification of 2-phenyl-2,3-dihydrophthalazine-1,4-dione (1), our model heterocyclic amide, as a follow-up to these studies. The single O-substituted result of the reaction of model ambient nucleophile 1 with ethyl chloroacetate in acetone in the presence of K2CO3under reflux for 12 h was (4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)acetic acid ethyl ester (2). The reaction was selectively carried out on the O atom rather than the N atom or even in a competitive reaction involving both atoms.

Calculating the interaction between HOMO on the oxygen atom of the surrounding high-energy nucleophile and the low-energy LUMO electrophile, which leads to a narrow energy gap and high reactivity, may explain the obtained chemoselective O-alkylation reaction. Pearson’s theory of hard soft acid base was used to arrive at this conclusion (HSAB). In ethanol, hydrazinolysis of ester 2 by refluxing with hydrazine hydrate for 8 h yields (4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetic acid hydrazide (3) in 72% yield, as shown in Scheme .

Scheme 1

Synthesis of Ester 2 and Corresponding Hydrazide 3 from Phthalazinedione

The characteristic 1H NMR spectral NCH2, NH (D2O exchangeable), and 9 aromatic protons gave signals at 4.32, 11.50, and 6.89–8.32 ppm for hydrazide 3. Hydrazide 3 is an excellent precursor for modifying the structure of subordinate phthalazines by using an azide strategy to attach another amino acid through a peptide bond. The azide strategy is a well-known peptide synthesis strategy that has the benefit of minimizing racemization while still avoiding any interferometer byproducts.

The azide 4 was formed by reacting hydrazide3 with a NaNO2/HCl mixture in water for 1 h at 0 °C and then extracting with ethyl acetate. To produce methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]alkanoates 5a–c, the in situ conveyed azide 4 solution was dynamically applied to amino acid methyl ester hydrochloride, glycine, β-alanine, and l-leucine within the sight of triethyl amine at 40 °C.

Hydrazinolysis of amino acid ester derivatives 5a–c in ethanol under reflux for 8 h with fabulous hydrazine hydrate. Scheme shows how to obtain the hydrazides 6a–c in high yield.

Scheme 2

Synthesis of Methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]alkanoate 5a–c and Their Corresponding Hydrazides 6a–c

1H NMR was used to elucidate the synthetic construction of methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]acetate (5a), which yielded the following signals: a broad signal at 6.91 ppm of NHCH2, a doublet signal at 4.07 ppm of NHCH2, and a singlet signal at 3.69 ppm of OCH3.

The phthalazines containing dipeptide moieties were also obtained using azide coupling methodology by reacting hydrazides 6a–c with the NaNO2/HCl blend and addition of amino acid methyl ester hydrochloride, glycine, β-alanine, and l-leucine in the presence of triethyl amine at 0 °C for 24 h to produce methyl-3-3-[2-(1,4-dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl)-acetylamino]propionyl-aminoalkanoates 7a–i, Scheme .

Scheme 3

Synthesis of Dipeptides 7a–i from Corresponding Hydrazides 6a–c

Different analysis methods were used to elucidate the structure of methyl 2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-acetylamino-acetate (7a). For example, the 1H NMR spectra of 7a revealed three singlet signals at 4.83, 4.00, and 3.87 ppm of three (−CH2) and a singlet signal at 3.65 ppm of OCH3.

Condensations of hydrazides 6a–c with various aldehydes in ethanol for 12 h under reflux conditions provide the corresponding Schiff’s bases 8a–i (Scheme ).

Scheme 4

Synthesis of Schiff’s Bases 8a–i from Corresponding Hydrazides 6a–c

Various investigation methods were used to clarify the structure, such as the 1H NMR spectra of N-[2-(4-chloro-benzylidene-hydrazinocarbonyl)-ethyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8a). The following signals appeared in −acetamide (8a): a broad signal of NH at 11.47 ppm, multiplet signal of 4H of the aromatic ring of chlorobenzaldhyde at 7.37–7.52 ppm, and singlet signal of CH at 10.11 ppm.

The phthalazine-containing monopeptide of 4-amino methylbenzoate moieties was also obtained using an azide coupling methodology by reacting hydrazide 3 with the NaNO2/HCl blend and adding 4-amino methylbenzoate at 0 °C for 24 h to produce methyl-4-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-benzoate (9) Scheme .

Scheme 5

Synthesis of Methyl-4-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-benzoate (9)

Biology

Cytotoxicity against HCT-116 and MDA-MB-231 Cell Lines

As VEGFR2 is one of the proteins that regulate the proliferation of HCT-116 and MDA-MB-231 cells, some of the synthesized compounds were screened for their cytotoxicity against HCT-116 and MDA-MB-231 cell lines using the MTT assay. Values of IC50 values were calculated, as shown in Table . Results indicated that the tested compounds, especially 7c and 8b with the phenyl phthalazinone moieties, had promising cytotoxicity against the HCT-116 cells with IC50 values of 1.36 and 2.34 μM, respectively. Compound 7c inhibited the proliferation of MDA-MB-231 cells with an IC50 value of 6.67 μM, while compound 8b exhibited moderate cytotoxicity with an IC50 value of 16.03 μM. Other tested compounds exhibited poor cytotoxic activities against the two tested cell lines. As seen in Figure , compounds 7c and 8b showed promising cytotoxicity, having cell viability by 10.36 and 11.69% at a concentration of 100 μM. Additionally, the promising compounds 7c and 8b exhibited poor cytotoxicity against WISH cells with much higher IC50 values, so they were safe against normal cells.

Table 1

IC50 (μM) of the Phenyl Phthalazinone Derivatives against HCT-116 and MDA-MB-231 Cells

	IC50 (μM)a
compound	HCT-116	MDA-MB-231
2	23.9	36.2
9	34.3	50≤
5c	26.39	12.36
6b	13.69	6.89
7c	1.36	7.67
7d	26.39	34.6
7e	16.39	36.9
7i	50≤	16.3
8a	6.34	27.3
8b	2.34	16.03
8i	13.5	24.3
cisplatin	3.67	5.71

Values are calculated using the GraphPad prism software using the non-linear regression curve fit; ND = not determined.

Figure 2

Cytotoxic activity of compounds 7c and 8b against HCT-116 and normal WISH cells. Upper panel: microscopy investigation after 48 h of incubation (40× magnification); lower panel: percentages of cell viabilities with increasing concentrations against the three tested cells.

Our results agreed with previous studies[9,10,19−22] that investigated novel phthalazine-based compounds against a panel of cancer cells; the studies proved the promising cytotoxic activities of such compounds with near IC50 values in a selective way. Additionally, they investigated the apoptotic cell death as the mechanism of action.

Anti-bacterial Activity

Results of anti-bacterial study are reported as the zone of inhibition and are summarized in Table , indicating that all tested compounds possess promising anti-bacterial activity against Staphylococcus aureus and Escherichia coli compared to the starting material 2-phenyl-2,3-dihydrophthalazine-1,4-dione 2. Compound 8c exhibited a potent anti-bacterial activity with inhibition zones of 12 and 11 mm against S. aureus and E. coli, respectively. Other compounds exhibited promising anti-bacterial activity with an inhibition zone of 6–10 mm. These findings supported the main aim of our work, which is to develop new potent anti-bacterial derivatives than the starting compound.

Table 2

Anti-bacterial Activity of the Compounds against S. aureus and E. colia

	Gram +ve	Gram −ve
compound	S. aureus NCMB6571	E. coli ATCC 25922
7a	7 ± 0.1	7 ± 0
7e	8 ± 0.2	7 ± 1.4
7h	9.5 ± 0.7	8 ± 1.4
chloroform	NA	NA
5a	8 ± 1.4	9 ± 0.1
8c	12 ± 1.4	11 ± 1.4
8d	8 ± 0.1	9 ± 0.1
8h	6 ± 0.1	10 ± 1.4
DEMSO	NA	NA
2-phenyl-2,3-dihydrophthalazine-1,4-dione 2	3 ± 0.1	NA

Values are Mean ± SD of measured inhibition zone diameter (mm) of two replicate. NA: Not Active.

In Silico Study

Molecular Docking

A molecular docking study was performed to confirm the binding interactions of the most active cytotoxic compounds 7c and 8b against the HCT-116 cells and explore their binding modes in conjunction with the co-crystallized ligands inside the active site VEGFR2 protein (1YWN). By visualization of the ligand–receptor interactions of the co-crystallized ligand (LIF), it was found that Glu 883, Asp 1044, and Cys 917 are the key amino acids with which it interacts. A molecular docking study revealed that both compounds 7c and 8b were effectively docked inside the protein active site with a good binding interactive mode like the co-crystallized ligand, as shown in the surface representation (Figure ). As seen in Table , with the summarized ligand–receptor interactions, compound 7c docked within the 1YWN with a high binding energy of −25.12 Kcal/mol, forming 3HB with Glu 883, Asp 1044, and Cys 917 as the main key amino acids with which the co-crystallized ligand (LIF) binds and one arene–arene with Lys 866. Meanwhile, compound 8b was docked with a good binding energy of −12.93 Kcal/mol, but it only formed 1HB with Glu 883.

Figure 3

Binding disposition of the co-crystallized ligand (orange-colored) and the docked compound (green-colored) of (A) 7c and (B) 8b toward the VEGFR2 protein (1YWN).

Table 3

Summarized Ligand–Receptor Interactions and 3D Visualization of the Two Docked Compounds 7c and 8b inside the VEGFR2 Protein (1YWN)a

Docking calculation using AutoDock4 software was validated by the self-docking step that fulfills RMSD lower than 2.

Thus, from the molecular docking results of compounds 7c and 8b, a good binding disposition and the ligand–receptor interactions like the co-crystallized ligand were maintained, and we may link their cytotoxic activities toward the VEGFR2 protein as the proposed mode of action.

ADME Pharmacokinetics

A bioinformatics study was conducted to establish the physicochemical properties and drug-like properties of the lead compounds 7c and 8b toward the tested proteins. The compounds examined were all well-permeable and absorbed. As shown in Table , compounds had two donors and six acceptors for hydrogen bonding. In addition, they exhibited log P values between 2.25 and 2.27, so they were well tolerated by cell membranes. For controlling conformational changes and oral bioavailability, the rotatable bond number (nrotb) should be ≤10. Additionally, compounds exhibited good gastrointestinal tract absorption according to the BOILED-Egg model, as shown in Figure .

Table 4

Molecular Properties and Drug-Likenessa

		compound
website	ADME	7c	8b
molinspiration 2018.10	Mwt (D)	480.5	485.5
	MV (A3)	432.63	424.7
	PSA (A2)	128.5	123.8
	log p	2.25	2.27
	nrotb	10	8
	nviolations	0	0
MolSoft	HBA	6	6
	HBD	2	2
	solubility (mg/L)	2000	1125
	drug-likeness score	0.27	0.48
SwissADME	drug-likeness (Lipinski Pfizer filter)	yes, drug like, MW ≤ 500, log p ≤ 4.15, HBA ≤ 10, and HDD ≤ 5

Mwt: molecular weight, MV: molecular volume, PAS: polar surface area, log p: octanol–water partition coefficient, nrotb: number of rotatable bond, nviolations: number of violations, HBA: hydrogen bond acceptor, HBD: hydrogen bond donor.

Figure 4

BOILED-Egg model for compounds (A) 7c and (B) 8b using the SwissADME. “Points located in the BOILED-Egg’s yolk are molecules predicted to passively permeate through the blood–brain barrier, while points located in the BOILED-Egg’s white are molecules predicted to be passively absorbed by gastrointestinal tract”.

Experimental Part

General Procedures

TLC was performed on silica gel 60 F254 aluminum sheets (E. Merck, layer thickness: 0.2 mm) in the preparation of dissolvable frameworks (S1: petroleum ether/ethyl acetate (2:1); S2: petroleum ether/ethyl acetic acid derivation (1:1)). The UV lamp recognized the spots on thin layer surfaces. The melting points were determined using a Buchi 510 melting-point system and are uncorrected. At the Micro Analytical Laboratory, Faculty of Science, Cairo University, Cairo, Egypt, elemental analyses were performed on a Flash EA-1112 apparatus. The nuclear magnetic resonance laboratory, Faculty of Science, Sohag University, Egypt, used a Bruker spectrometer running at 400 MHz to estimate the 1H NMR spectra. The phthalazinediones were made from the precursor 2-phenyl-2,3-dihydrophthalazine-1,4-dione (1) according to the method described by Gildeh et al.[23]

Synthesis of Ethyl (1,4-Dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl)acetate (2)

Ethyl chloroacetate (12.25 mL, 0.1 mol) and 1.2 equiv of K2CO3 were applied to a solution of 2-phenyl-2,3-dihydrophthalazine-1,4-dione (1) (23.8 g, 0.1 mol) in 50 mL of dry acetone and 10 mL of DMF mixture. Overnight, the reaction mixture was refluxed. The clear reaction mixture solution was poured over crushed ice, and the solid product obtained was filtered and washed with water several times and was crystallized from aq. EtOH to obtain pure ethyl-3-(1,4-dioxo-3-phenyl-3,4-dihydrophthalazin-2(1H)-yl)-2-oxopropanoate(2). Off-white, crystals yield (78%), m.p.166–164 °C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz):8.38–8.41 (2H, m, ArH);8.00–8.02 (2H, m, ArH);7.71–7.77 (2H, m, ArH);7.61 (1H, d, J = 8, ArH);7.17–7.38 (2H, m, ArH);4.82 (2H, s, CH2); 4.17 (2H, q, J = 7.1, CH2CH3);1.16 (3H, t, J = 7.1, CH2CH3).MS. (MALDI, positive mode, matrix DHB) m/z: 347 (M + Na)+. Anal. Calcd. for C18H16N2O4(324.3): C, 66.66; H, 4.97; N, 8.64. Found: C, 66.74; H, 5.06; N, 8.73.

Synthesis of (4-Oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetic Acid Hydrazide (3)

In 25 mL of ethanol, a mixture of ester 2 (3.24 g, 0.01 mol) and hydrazine hydrate (5 mL, 99%) was heated under reflux for 8 h. The resulting precipitate was cooled, then purified, and crystallized from MeOH to yield the hydrazide 3. White fine powder (72%) with a melting point of 262–264 °C.(400 MHz, CDCl3), δ, ppm (J, Hz): 9.4 (1H, S, NH); 8.21–8.34 (2H, m, ArH); 7.95–8.1 (2H, m, ArH); 7.70–7.73 (2H, m, ArH); 7.48–7.52 (1H, m,ArH); 7.35–7.39 (2H, m, ArH); 4.81 (2H, s, CH2); 4.35 (2H, s, NH2). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 65.0 (OCH2), 124.3, 124.5, 125.6 (2C), 127.38, 127.41 (2C), 128.9, 129.4, 133.3, 134.1, 142.1, 149.1, 157.9 (C=O), 166.6 (C=O). MS. (MALDI, positive mode, matrix DHB) m/z: 334 (M + Na)+. Anal. Calcd. for C16H14N4O3 (310.3) C, 61.93; H, 4.55; N, 18.06. Found: C, 62.01; H, 4.63; N, 18.14.

General Procedure for Preparation of Methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]alkanoate 5a–c

Hydrochloric acid (5 N, 30 mL), included parcel shrewd under blending a cold solution (0 °C) of sodium nitrite (0.7 g, 10.0 mmol) in water (30 mL) to a cold solution (− 5 °C) of hydrazide 3 (8.0 mmol) in acetic acid (60 mL) at the same temperature for 30 min. The in situ created azide 4a–c was extricated with cold ethyl acetate and washed gradually with cold water, 5% NaHCO3, and water. The azide was used without further purification in the next step after drying over anhydrous sodium sulfate. Amino acid methyl ester hydrochloride (9.0 mmol), glycine, β-alanine, and l-leucine were added to a cold dried solution of azide 4a–c that had already been prepared. After that, the mixture was kept in the fridge for 12 h and then at room temperature for another 12 h. The reaction mixture was washed with 0.1N HCl, water, 5% NaHCO3, and water and then dried over anhydrous sodium sulfate, the solvent was evaporated in vacuum, and the buildup was crystallized from ethyl acetate-petroleum ether to yield the products 5a–c.

Methyl [2-(4-Oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-acetate (5a)

Off-white crystals (77%), m.p. 118–119 °C (400 MHz, CDCl3), δ, ppm (J, Hz): 8.42–8.39 (1H, m, ArH); 7.98–7.96 (1H, m, ArH); 7.80–7.75 (2H, m, ArH); 7.63 (2H, d, J = 8.1, ArH); 7.37–7.35 (2H, m, ArH); 7.24–7.22 (1H, m, ArH); 6.91 (1H, bs, NH); 4.84 (2H, s, CH2); 4.06 (2H, d, J = 6.9, NHCH2); 3.69 (3H, s, OCH3). MS. (MALDI, positive mode, matrix DHB) m/z: 390 (M + Na)+. Anal. Calcd. for C19H17N3O5 (367.3) C, 62.12; H, 4.66; N, 11.44. Found: C, 62.30; H, 4.84; N, 11.63.

Methyl-3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-propionate (5b)

Off-white crystals (81%), m.p. 114–115 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 8.42 (2H, d, J = 8.0, ArH); 7.9 (2H, d, J = 8.1, ArH); 7.63–7.83 (2H, m, ArH); 7.19–7.40 (3H, m, ArH); 6.87 (1H, bs, NH); 4.78 (2H, s, CH2); 3.57 (3H, s, OCH3); 3.31–3.29 (2H, m, NHCH2); 2.20–2.24 (2H, m, CH2CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Anal. Calcd. for C20H19N3O5 (381.3): C, 62.99; H, 5.02; N, 11.02. Found: C, 63.07; H, 5.14; N, 11.11.

Methyl-4-methyl-2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-pentanoate (5c)

Off-white crystals (72%), m.p. 122–123 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 8.42–8.45 (2H, m, ArH); 7.96–7.98 (2H, m, ArH); 7.75–7.83 (2H, m, ArH); 7.63–7.65 (1H, m, ArH); 7.19–7.39 (2H, m, ArH); 6.87 (1H, bs, NH); 4.85 (2H, s, CH2); 4.67 (1H, q, CH); 3.65 (3H, s, OCH3); 1.74–172 (2H, m, CH2); 1.55(1H, m, (CH3)2CH); 0.83 (6H, d, (CH3)2CH). MS. (MALDI, positive mode, matrix DHB) m/z: 446 (M + Na)+. Anal. Calcd. for C23H25N3O5 (423.46): C, 65.24; H, 5.95; N, 9.92. Found: C, 65.43; H, 6.14; N, 10.10.

Synthesis of Hydrazides 6a–c

Hydrazine hydrate was added to a solution of ester 5a–c (3.67 g, 0.01 mol) in methyl alcohol (30 mL) (2.5 mL, 0.05 mol). To obtain the corresponding hydrazide, the reaction mixture was refluxed for 4 h and cooled, and the white precipitate was purified and crystallized from MeOH.

Synthesis of N-Hydrazinocarbonylmethyl-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (6a)

Off-white crystals (87%), m.p. 193–195 °C. 1H NMR(400 MHz, DMSO), δ, ppm (J, Hz): 9.4 (1H, bs, NH); 8.34–8.37 (2H, m, ArH); 8.21–8.23 (2H, m, ArH); 7.97–8.04 (2H, m, ArH); 7.71–7.73 (1H, m, ArH); 7.35–7.52 (2H, m, ArH); 6.32 (1H, bs, NH); 4.79 (2H, s, CH2); 4.02 (2H, d, J = 6.9, NHCH2); 4.47 (2H, bs, NH2). MS. (MALDI, positive mode, matrix DHB) m/z: 390 (M + Na)+. Anal. Calcd. for C18H17N5O4 (367.4): C, 58.85; H: 4.66; N: 19.06. Found: C, 59.06; H, 4.84; N, 19.12.

Synthesis of N-(2-Hydrazinocarbonyl-ethyl)-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (6b)

Off-white crystals (84%), m.p. 186–188 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 9.02 (1H, bs, NH); 8.34–8.36 (2H, d, J = 8.2, ArH); 8.21 (2H, d, J = 8.0, ArH); 7.95–8.05 (2H, m, ArH); 7.69–7.71 (2H, m, ArH); 7.45–7.52 (1H, m, ArH); 6.02 (1H, bs, NH); 4.77 (2H, s, CH2); 4.51 (2H, bs, NH2); 3.4 (2H, m, CH2); 2.26 (2H, m, CH2). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 33.3 (CH2), 35.7 (CH2), 65.7 (OCH2), 124.3, 125.6, 127.4, 128.9, 129.4, 133.3, 134.2, 142.1, 149.1, 157.9 (C=O), 167.3 (C=O), 170.2 (C=O). MS. (MALDI, positive mode, matrix DHB) m/z: 404 (M + Na)+. Anal. Calcd. for C19H19N5O4 (381.4): C; 59.84, H; 5.02, N; 18.36. Found: C, 59.93; H, 5.11; N, 18.44.

Synthesis of N-(1-Hydrazinocarbonyl-3-methyl-butyl)-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (6c)

Off-white crystals (88%), m.p. 198 °C. 1H NMR (400 MHz, CDCl3): 9.19 (1H, bs, NH); 8.35 (2H, d, J = 8.1, ArH); 8.19–8.11 (2H, m, ArH); 8.04–7.97 (2H, m, ArH); 7.71 (2H, d, J = 8.0, ArH); 7.36–7.50 (1H, m, ArH); 6.22 (1H, bs, NH); 4.88–4.86 (1H, m, CH); 4.51–4.49 (2H, m, CH2); 4.29 (2H, bs, NH2); 1.94–1.91 (2H, m, CH2); 1.45–1.43 (1H, m, (CH3)2CH); 0.80 (6H, d, J = 7.0, (CH3)2CH)). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 22.0, 23.3, 24.6, 65.5 (OCH2), 124.2, 124.3, 125.5, 127.3, 127.5, 128.8, 129.4, 133.3, 134.3, 142.1, 149.2, 157.9 (C=O), 167.1 (C=O), 171.3 (C=O). MS. (MALDI, positive mode, matrix DHB) m/z: 446 (M + Na)+. Anal. Calcd. for C22H25N5O4 (423.5): C, 62.40; H, 5.95; N, 16.54. Found: C, 62.49; H, 6.04; N, 16.63.

General Procedures for Preparation of Dipeptide 7a–i

A solution of NaNO2 (0.7 g, 0.01 mol) in cold water (15 mL) was added to a cold solution (0 °C) of hydrazide (6) (3.67 g, 0.01 mol) in acetic acid (15 mL), 1N HCl (15 mL), and water (25 mL). The reaction mixture was blended for 15 min at 0 °C. Cold ethyl acetate (30 mL) was used to extract the yellow syrup, which was then washed with cold 5% Na2CO3 (30 mL) and dried over Na2SO4. The amino acid ester NH2(CHR)COOMe·HCl (0.01 mol) in ethyl acetate (20 mL) with 2 mL of Et3N was added to this solution. The reaction mixture was held at 0 °C for 24 h before being moved to room temperature for another 24 h. The solution evaporated into nothingness. To achieve the desired result, the buildup was crystallized from petroleum ether/ethyl acetate.

Methyl {2-[2-(4-Oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-acetylamino}-acetate (7a)

From hydrazide 6a (3.67 g, 0.01 mol) and glyOCH3·HCl (1.25 g, 0.01 mol)

Off-white crystals (76%), m.p. 144–143 °C. 1H NMR(400 MHz, CDCl3), δ, ppm (J, Hz): 10.64 (1H, bs, NH); 8.40 (2H, d, J = 8.10, ArH); 7.62–7.79 (2H, m, ArH); 6.68–6.91 (2H, m, ArH); 6.88 (3H, m, ArH); 6.21 (1H, bs, NH); 4.83 (2H, s, CH2); 4.00 (2H, s, CH2); 3.87 (2H, s, CH2); 3.65 (3H, s, OCH3). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 41.1, 42.6, 52.4, 65.6 (OCH2), 123.2, 123.8, 124.9, 127.2, 128.0, 128.6, 129.9, 132.6, 133.5, 141.6, 148.5, 158.3 (C=O), 168.1 (C=O), 168.6 (C=O), 169.9 (C=O).MS. (MALDI, positive mode, matrix DHB) m/z: 447 (M + Na)+. Anal. Calcd. for C21H20N4O6 (424.4): C, 59.43; H, 4.75; N, 13.20. Found: C, 59.62; H, 4.90; N, 13.38.

Methyl-3-{2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-acetylamino}-propionate (7b)

From hydrazide 6a (3.67 g, 0.01 mol) and β-AlaOCH3·HCl (1.4 g, 0.01 mol)

Off-white crystals (79%), m.p. 137–136 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 10.64 (1H, bs, NH); 8.40 (2H, d, J = 8.1, ArH); 8.0 (2H, d, J = 8.0, ArH); 7.62–7.09 (2H, m, ArH); 7.19–7.39 (1H, m, ArH); 6.72–6.90 (2H, m, ArH); 6.69 (1H, bs, NH); 4.82 (2H, s, CH2); 4.12 (2H, s, CH2); 3.92 (2H, m, CH2); 3.58 (3H, s, OCH3); 2.64 (2H, m, CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 461 (M + Na)+. Anal. Calcd. for C22H22N4O6 (438.4): C, 60.27; H, 5.06; N, 12.78. Found: C, 60.44; H, 5.20; N, 12.91.

Methyl-4-methyl-2-{2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-acetylamino}-pentanoate (7c)

From hydrazide (6a) (3.67 g, 0.01 mol) and l-leuOCH3·HCl (1.81 g, 0.01 mol)

Off-white crystals (73%), m.p. 129–130 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 10.55 (1H, bs, NH); 8.42 (2H, d, J = 8.0, ArH); 8.1 (2H, d, J = 7.9, ArH); 7.62–7.81 (2H, m, ArH); 7.29–7.39 (1H, m, ArH); 6.64–6.91 (2H, m, ArH); 6.25 (1H, bs, NH); 4.83–4.81 (1H, m, CH); 4.79 (2H, s, CH2); 3.87 (2H, s, CH2); 3.65–3.64 (2H, m, CH2); 3.52 (3H, s, OCH3); 1.57(1H, m, CH-(CH3)2); 0.85(6H, d, CH-(CH3)2). MS. (MALDI, positive mode, matrix DHB) m/z: 461 (M + Na)+. Anal. Calcd. for C25H28N4O6 (480.5): C, 62.49; H, 5.87; N, 11.66. Found: C, 62.55; H, 6.13; N, 11.75.

Methyl-2{3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-propionylamino}-acetate (7d)

From hydrazide 6b (3.81 g, 0.01 mol) and glyOCH3·HCl (1.25 g, 0.01 mol)

Off-white crystals (77%), m.p. 134–135 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz):8.89 (1H, bs, NH); 8.51 (2H, d, J = 8.0, ArH); 8.15 (2H, d, J = 7.9, ArH); 7.84–7.92 (2H, m, ArH); 7.74–7.76 (1H, m, ArH); 7.28–7.50 (2H, m, ArH); 6.25 (1H, bs, NH); 4.88 (2H, s, CH2); 3.97–3.95 (2H, m, CH2); 3.70(3H, s, OCH3); 3.44–3.42 (2H, m, CH2); 2.37–2.35 (2H, m, CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 461 (M + Na)+. Anal. Calcd. for C22H22N4O6 (438.4): C, 60.27; H, 5.06; N, 12.78. Found: C, 60.36; H, 5.21; N, 12.84.

Methyl-3-{3-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-propionylamino}-propionate (7e)

From hydrazide 6b (3.81 g, 0.01 mol) and β-alaOCH3·HCl (1.4 g, 0.01 mol)

Off-white crystals (81%), m.p.129–130 °C. 1H NMR (400 MHz, CDCl3): 9.09 (1H, bs, NH); 8.49 (2H, d, J = 8.0, ArH); 8.16 (2H, d, J = 8.2, ArH); 7.83–7.91 (2H, m, ArH); 7.72–7.74 (2H, m, ArH); 7.29–7.48 (1H, m, ArH); 6.19 (1H, bs, NH); 4.85(2H, s, CH2); 3.76 (3H, s, OCH3); 3.63–3.61 (2H, m, CH2); 3.43–3.41 (2H, m, CH2); 2.50–2.51 (2H, m, CH2); 2.40–2.38 (2H, m, CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 475 (M + Na)+. Anal. Calcd. for C23H24N4O6 (452.5): C, 61.05; H, 5.35; N, 12.38. Found: C, 61.14; H, 5.45; N, 12.42.

Methyl {4-Methyl-2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-pentanoyl} Acetate (7f)

From hydrazide 6b (3.81 g, 0.01 mol) and l-leuOCH3·HCl (1.81 g, 0.01 mol)

Off-white crystals (83%), m.p. 124–125 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 9.12 (1H, bs, NH); 8.41 (2H, d, J = 8.0, ArH); 8.08 (2H, d, J = 8.1, ArH); 7.74–7.81 (2H, m, ArH); 7.64–7.66 (2H, m, ArH), 7.29–7.48 (1H, m, ArH), 6.19 (1H, bs, NH); 4.78 (2H, s, CH2); 4.51–4.49 (1H, m, CH); 3.32–3.31 (2H, m, CH2); 2.65–2.64 (2H, m, CH2); 3.58 (3H, s, OCH3); 2.42–2.40 (2H, m, CH2); 1.55–1.54 (1H, m, (CH3)2CH); 0.85–0.83 (6H, m, (CH3)2CH). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 21.8, 22.0, 22.7, 24.9, 35.1, 35.2, 41.4, 50.9, 52.3, 65.5 (OCH2), 123.5, 124.0, 125.0, 127.2, 127.9, 128.6, 129.8, 132.4, 133.4, 141.7, 148.6, 158.4 (C=O), 167.7 (C=O), 171.7 (C=O), 173.3 (C=O). MS. (MALDI, positive mode, matrix DHB) m/z: 517 (M + Na)+. Anal. Calcd. for C26H30N4O6 (494.5): C, 63.15; H, 6.11; N, 11.33. Found: C, 63.22; H, 6.20; N, 11.40.

Methyl {4-Methyl-2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-pentanoylamino}-acetate (7g)

From hydrazide 6c (4.23 g, 0.01 mol) and glyOCH3·HCl (1.25 g, 0.01 mol)

Off-white crystals (75%), m.p. 127 °C. 1H NMR(400 MHz, CDCl3), δ, ppm (J, Hz): 8.50 (2H, d, J = 8.0, ArH); 8.05 (2H, d, J = 8.1, ArH); 7.83–7.90 (2H, m, ArH); 7.71–7.73(2H, m, ArH); 7.28–7.48 (2H, m, ArH & NH); 6.20 (1H, bs, NH); 4.88–4.86 (1H, m, CH); 4.24 (2H, s, CH2); 3.94 (2H, d, J = 7.0, CH2); 3.72 (3H, s, OCH3); 1.94–1.92 (2H, m, CH2); 1.63 (1H, m, (CH3)2CH); 0.09 (6H, d, J = 6.9, (CH3)2CH). MS. (MALDI, positive mode, matrix DHB) m/z: 503 (M + Na)+. Anal. Calcd. for C25H28N4O6 (480.5): C, 62.49; H, 5.87; N, 11.66. Found: C, 62.58; H, 6.00; N, 11.72.

Methyl-3-{4-Methyl-2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-pentanoylamino}-propionate (7h)

From hydrazide 6c (4.23 g, 0.01 mol) and β-alaninOCH3·HCl (1.4 g, 0.01 mol)

Off-white crystals (81%), m.p. 123–122 °C. 1H NMR (400 MHz, CDCl3), δ, ppm (J, Hz): 8.51 (2H, d, J = 8.0, ArH); 8.06 (2H, d, J = 8.2, ArH); 7.83–7.91 (2H, m, ArH); 7.71–7.73 (2H, m, ArH & NH);7.28–7.47 (2H, m, ArH); 6.11 (1H, bs, NH); 4.90(1H, m, CHCH2); 3.55–3.53 (2H, m, CH2); 3.68 (3H, s, OCH3); 3.54–3.52 (2H, m, CH2); 2.56–2.54 (2H, m, CH2); 2.52 (2H, m, CH2); 1.60 (1H, m, (CH3)2CH); 0.89 (6H, d, J = 6.9, (CH3)2CH). MS. (MALDI, positive mode, matrix DHB) m/z: 517 (M + Na)+. Anal. Calcd. for C26H30N4O6 (494.5) C, 63.15; H, 6.11; N, 11.33. Found: C, 63.34; H, 6.30; N, 11.52.

Methyl-4-methyl-2-{4-methyl-2-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-pentanoylamino}pentanoate (7i)

From hydrazide 6c (4.23 g, 0.01 mol) and l-leuOCH3·HCl (1.81 g, 0.01 mol)

Off-white crystals (78%), m.p. 119–118 °C. 1H NMR(400 MHz, CDCl3), δ, ppm (J, Hz): 8.51 (2H, d, J = 8.0, ArH); 8.05 (2H, d, J = 7.9, ArH); 7.83–7.90 (2H, m, ArH); 7.71–7.73 (2H, m, ArH&NH); 7.28–7.47 (2H, m, ArH); 6.18 (1H, bs, NH); 4.90–4.88 (1H, m, CHCH2); 4.65–4.64 (1H, m, CHCH2); 4.20 (2H, s, CH2); 3.73 (3H, s, OCH3); 1.97–1.95 (2H, m, CH2); 1.91–1.92 (1H, m, (CH3)2CH); 1.77–1.75 (2H, m, CH2); 1.62–1.60 (1H, m, (CH3)2CH); 0.93 (6H, d, J = 7.0, (CH3)2CH); 0.88 (6H, d, J = 6.9, (CH3)2CH). MS. (MALDI, positive mode, matrix DHB) m/z: 559 (M + Na)+. Anal. Calcd. for C29H36N4O6 (536.6): C, 64.91; H, 6.76; N, 10.44. Found: C, 65.02; H, 6.85; N, 10.49.

General Procedures for Preparation of Hydrazones 8a–i

A mixture of one of the hydrazides 6a–c (0.01 mol) and an effective aromatic aldehyde (0.01 mol), such as 4-chloro benzaldehyde, anisaldehyde, and/or 4-nitro benzaldehyde, was refluxed in ethanol (25 mL) for 12 h and then crystallized from the ethanol.

Synthesis of N-(4-Chloro-benzylidene-hydrazinocarbonylmethyl)-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8a)

From 4-chloro-benzaldehyde and hydrazide 6a

Off-white crystals (84%), m.p. 187–188 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 11.47 (1H, bs, NH); 8.38 (2H, d, J = 8.0, ArH); 8.23 (2H, d, J = 8.2, ArH);7.98–8.03 (2H, m, ArH); 7.69–7.77 (3H, m, ArH); 7.37–7.52 (4H, m, ArH); 6.69 (1H, s, =CH); 6.04 (1H, bs, NH); 4.90(2H, s,CH2); 4.35(2H, d, J = 7.0,CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 513 (M + Na)+. Anal. Calcd. for C25H20ClN5O4 (489.9): C, 61.29; H, 4.11;Cl, 7.24; N, 14.30. Found: C, 61.38; H, 4.20;Cl, 7.33; N, 14.39.

Synthesis of N-(4-Methoxy-benzylidene-hydrazinocarbonylmethyl)-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8b)

From 4-methoxy-benzaldehyde and hydrazide 6a

Off-white crystals (78%), m.p. 181–182 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 11.35 (1H, bs, NH); 8.36 (2H, d, J = 8.0, ArH); 8.24 (2H, d, ArH, J = 8.1); 7.95–8.04 (2H, m, ArH); 7.87–7.89 (3H, m, ArH); 7.48–7.77 (2H, m, ArH); 7.12–7.14 (2H, m, ArH); 6.58 (1H, s, =CH); 6.12 (1H, bs, NH); 4.90(2H, s, CH2); 4.44 (2H, d, CH2); 3.88 (3H,s, OCH3). MS. (MALDI, positive mode, matrix DHB) m/z: 508 (M + Na)+. Anal. Calcd. for C26H23N5O5 (485.5): C, 64.32; H, 4.78; N, 14.43. Found: C, 64.41; H, 4.86; N, 14.52.

Synthesis of N-(4-Nitro-benzylidene-hydrazinocarbonylmethyl)-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8c)

From 4-nitro-benzaldehyde and hydrazide 6a

Off-white crystals (78%), m.p. 191–192 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 11.82 (1H, bs, NH); 8.35 (2H, d, J = 8.0, ArH); 8.25 (2H, d, J = 8.1, ArH); 7.93–8.03 (2H, m, ArH); 7.74–7.76 (3H, m, ArH); 7.37–7.52 (4H, m, ArH); 6.81 (1H, s, =CH); 6.11 (1H, bs, NH); 4.91 (2H, s, CH2); 4.39 (2H, d, J = 7.0, CH2). MS. (MALDI, positive mode, matrix DHB) m/z: 523 (M + Na)+. Anal. Calcd. for C25H20N6O6 (500.5): C, 60.00; H, 4.03; N, 16.79. Found: C, 60.12; H, 4.08; N, 16.88.

Synthesis of N-[2-(4-Chloro-benzylidene-hydrazinocarbonyl)-ethyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8d)

From 4-chloro-benzaldehyde and hydrazide 6b

Off-white crystals (80%), m.p. 194–195 °C. 1H NMR (400 MHz, DMSO): 11.42 (1H, bs, NH); 8.34 (2H, d, J = 8.2, ArH); 8.16–8.23 (2H, m, ArH); 7.95–8.04 (4H, m, ArH); 7.63–7.70 (2H, m, ArH); 7.41–7.51 (1H, m, ArH); 7.33 (2H, d, J = 8.2, ArH); 6.77 (1H, s, =CH); 6.08 (1H, bs, NH); 4.78(2H, s, CH2); 3.45–3.44 (1H, m, CH); 2.22 (3H, d, J = 7.0, CH3). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 32.8, 34.9, 65.7 (OCH2), 124.3, 125.5, 127.4, 127.5, 128.7, 129.1, 129.4, 133.3, 133.6, 134.6, 142.1, 145.4, 149.0, 157.9 (C=O), 167.4 (C=O), 167.6 (C=O), 173.3 (C=O). MS. (MALDI, positive mode, matrix DHB) m/z: 527 (M + Na)+. Anal. Calcd. for C26H22ClN5O4 (503.9): C, 61.97; H, 4.40;Cl, 7.04; N, 13.90. Found: C, 62.06; H, 4.48;Cl, 7.13; N, 14.01.

Synthesis of N-[2-(4-Methoxy-benzylidene-hydrazinocarbonyl)-ethyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8e)

From 4-methoxy-benzaldehyde and hydrazide 6b

Off-white crystals (82%), m.p. 190–191 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 9.92 (1H, bs, NH); 8.56 (2H, d, J = 8.2, ArH); 7.86–7.92 (2H, m, ArH); 7.72 (2H, d, J = 8.2, ArH); 7.42–7.59 (3H, m, ArH); 7.02–7.04 (2H, d, J = 8.2, ArH); 7.92–7.94 (2H, d, J = 8.2, ArH); 6.68 (1H, s, =CH); 6.06 (1H, bs, NH); 4.92 (2H, s, CH2); 3.92 (1H, s, CH); 3.87 (3H, s OCH3); 1.54 (3H, d, CH3). MS. (MALDI, positive mode, matrix DHB) m/z: 522 (M + Na)+. Anal. Calcd. for C27H25N5O5 (499.5): C, 64.92; H, 5.04; N, 14.02. Found: C, 65.00; H, 5.11; N, 14.14.

Synthesis of N-[2-(4-Nitro-benzylidene-hydrazinocarbonyl)-ethyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8f)

From 4-nitro-benzaldehyde and hydrazide 6b

Off-white crystals (88%), m.p. 201–202 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 11.63 (1H, bs, NH); 8.32–8.34 (2H, m, ArH); 8.18–8.28 (2H, m, ArH); 7.85–8.07 (4H, m, ArH); 7.70 (2H, d, J = 8.0, ArH); 7.77–7.48 (1H, m, ArH);7.29–7.37 (2H, m, ArH); 6.57 (1H, s, =CH); 6.10 (1H, bs, NH); 4.77 (2H, s, CH2); 3.26 (1H, m, CH); 2.89 (3H, d, J = 7.0, CH3). MS. MALDI, positive mode, matrix DHB) m/z: 537 (M + Na)+. Anal. Calcd. for C26H22N6O6 (514.5): C, 60.70; H, 4.31; N, 16.33. Found: C, 60.78; H, 4.40; N, 16.42.

Synthesis of N-[1-(4-Chloro-benzylidene-hydrazinocarbonyl)-3-methyl-butyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8g)

From 4-chloro-benzaldehyde and hydrazide 6c

Off-white crystals (81%), m.p. 190–191 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 11.55 (1H, bs, NH); 8.40 (2H, d, J = 8.1, ArH); 8.25 (2H, d, J = 8.0, ArH); 8.12 (2H, d, J = 8.2, ArH); 7.95–8.01 (2H, m, ArH); 7.64–7.72 (2H, m, ArH); 7.46 (1H, d, J = 8.2,ArH); 7.32–7.37 (2H, m, ArH); 6.66 (1H, s, =CH); 6.12 (1H, bs, NH); 4.89–4.88 (1H, m, CH); 4.48–4.47 (2H, m, CH2); 1.94–1.91 (2H, m, CH2); 1.41–1.39 (1H, m, (CH3)2CH); 0.83 (6H, d, J = 7.0, (CH3)2CH). MS. (MALDI, positive mode, matrix DHB) m/z: 569 (M + Na)+. Anal. Calcd. for C29H28ClN5O4 (546.0): C, 63.79; H, 5.17; Cl, 6.49; N, 12.83. Found: C, 63.98; H, 5.36;Cl, 6.68; N, 13.00.

Synthesis of N-[1-(4-Methoxy-benzylidene-hydrazinocarbonyl)-3-methyl-butyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8h)

From 4-methoxy-benzaldehyde and hydrazide 6c

Off-white crystals (82%), m.p. 200–201 °C. 1H NMR (400 MHz, DMSO), δ, ppm (J, Hz): 10.44 (1H, bs, NH); 8.50 (2H, d, J = 8.0, ArH); 8.07–8.11 (2H, m, ArH); 7.41–7.88 (2H, m, ArH); 7.28–7.34 (2H, m, ArH); 7.18 (1H, d, J = 8.0, ArH); 6.86–6.91(4H, m, ArH); 6.66 (1H, s, =CH); 6.12 (1H, bs, NH); 4.89–4.87 (1H, m, CH); 4.52–4.50 (2H, m, CH2); 3.84(3H, s, OCH3); 1.90–1.89 (2H, m, CH2); 1.70–1.69 (1H, m, (CH3)2CH); 1.06 (6H, d, J = 7.0, (CH3)2CH).MS. (MALDI, positive mode, matrix DHB) m/z: 564.8 (M + Na)+. Anal. Calcd. for C30H31N5O5 (541.6): C, 66.53; H, 5.77; N, 12.93. Found: C, 66.62; H, 5.86; N, 12.98.

Synthesis of N-[3-Methyl-1-(4-nitro-benzylidene-hydrazinocarbonyl)-butyl]-2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetamide (8i)

From 4-nitro-benzaldehyde and hydrazide 6c

Off-white crystals (79%), m.p. 205–206 °C. 1H NMR(400 MHz, DMSO), δ, ppm (J, Hz): 11.79 (1H, bs, NH); 8.22–8.41 (4H, m, ArH); 7.87–8.14 (4H, m, ArH);7.71 (2H, d, J = 8.1, ArH); 7.45–7.49 (2H, m, ArH); 7.33–7.38 (1H, m, ArH); 6.71 (1H, s, =CH); 6.09 (1H, bs, NH); 4.90–4.88 (1H, m, CH); 4.55–4.53 (2H, m, CH2); 1.89–1.87 (2H, m, CH2); 1.41–1.39 (1H, m, (CH3)2CH); 0.86 (6H, d, J = 7.0, (CH3)2CH). Ms. (MALDI, positive mode, matrix DHB) m/z: 579.9 (M + Na)+. Anal. Calcd. for C29H28N6O6 (556.6): C, 62.58; H, 5.07; N, 15.10. Found: C, 62.66; H, 5.15; N, 15.19.

Synthesis of Methyl-4-[2-(4-oxo-3-phenyl-3,4-dihydro-phthalazin-1-yloxy)-acetylamino]-benzoate (9)

A solution of NaNO2 (0.7 g, 0.01 mol) in cold water (15 mL)was added to a cold solution (0 °C) of hydrazide (6) (3.67 g, 0.01 mol) in acetic acid (15 mL), 1N HCl (15 mL), and water (25 mL). The reaction mixture was blended for 15 min at 0 °C. Cold ethyl acetate (30 mL) was used to extract the yellow syrup, which was then washed with cold 5% Na2CO3 (30 mL) and dried over Na2SO4. The methyl-4-aminobenzoate (0.01 mol) in ethyl acetate (20 mL) with 2 mL of Et3N was added to this solution. The reaction mixture was held at 0 °C for 24 h before being moved to room temperature for another 24 h. The solution evaporated into nothingness. To achieve the desired result, the buildup was crystallized from petroleum ether/ethyl acetate.

Off-white crystals (68%), m.p. 115–116 °C. 1H NMR(400 MHz, CDCl3), δ, ppm (J, Hz): 8.47 (2H, d, J = 8.10, ArH); 7.95–8.09 (4H, m, ArH); 7.83 (2H, d, J = 8.0, ArH); 7.54–7.62 (2H, m, ArH); 7.27–7.39 (1H, m, ArH); 7.17 (2H, d, J = 8.20, ArH); 6.82 (1H, bs, NH); 4.95 (2H, s, CH2); 3.80 (3H, s, OCH3). 13C NMR (100.0 MHz, CDCl3), δ, ppm: 52.0, 66.2 (OCH2), 125.0, 126.5, 127.4, 128.3, 128.7, 130.1, 130.9, 132.8, 133.5, 140.9, 141.5, 148.3, 158.2, (C=O), 165.7(C=O), 166.4(C=O). Ms. (MALDI, positive mode, matrix DHB) m/z: 452 (M + Na)+. Anal. Calcd. for C24H19N3O5 (429.4): C, 67.13; H, 4.46; N, 9.79. Found: C, 67.22; H, 4.45; N, 9.87.

Biological Investigation

Cytotoxic Activity against HCT-116 and MDA-MB-231 Cell Lines

The MTT assay was used to examine the cytotoxic activity of the tested compounds against two cancer cell lines: colon (HCT-116) and breast (MDA-MB-231). The cells grew after being cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, HEPES buffer, and 50 g/mL gentamycin. Cells were subcultured and kept at 37 °C in a humidified atmosphere with 5% CO2 according to routine tissue culture work. The association between surviving cells and drug concentration is used to plot the survival curve of each tumor cell line after treatment with the relevant substance. The 50% inhibitory concentration (IC50), or the concentration required to cause cytotoxicity effects in 50% of cells, was derived using practical dose response curve graphs for each concentration using the GraphPad Prism software (San Diego, CA. USA).[24−26]

Anti-bacterial Activity

Tested compounds were evaluated for their anti-bacterial activities using “disc diffusion method”1 against the indicator strains E. coli (ATCC 25922) and S. aureus (NCMB6571) at two concentrations of samples (0.25 mg/disc & 0.75 mg/disc and 30 μg each). DMSO was used as a solvent and negative control. After the incubation period, the growth inhibition zones diameter were carefully measured in millimeters.[27]

Molecular Docking Studies

A molecular docking study was performed on a computational software basis using the (AutoDock4 2016-08 Chemical Computing Group, Canada) toward the Protein Data Bank’s X-ray crystal structure of VEGFR2 in complex with a novel 4-amino-furo[2,3-d]pyrimidine (PDB ID: 1YWN). Principles of modeling regarding receptor and ligand preparation and molecular docking were carried out according to Nafie et al..[28] Each ligand–receptor complex was tested for binding energy (Kcal/mol), interaction analysis, and 3D images, which were taken by Chimera as a visualizing software.

ADME Pharmacokinetics

In silico ADME pharmacokinetics parameters of the lead compounds were calculated using a set of software including “MolSoft”, “Molinspiration”, and “SwissADME” websites as previously described.[29,30]

Conclusions

Searching for novel target-oriented anti-cancer agents is still a continuous research process to investigate effective and selective chemotherapeutic agents. Here, in the study, there are 18 new phthalazinediones beginning with methyl-1,4-dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl-acetate (2) synthesized via O-alkylation of 2-phenyl-2,3-dihydrophthalazine-1,4-dione (1) with ethyl chloroacetate, yielding the starting material 2.3-[2-(1,4-dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl)-acetylamino] monopeptide methyl-3-[2-(1,4-dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl)acetylamino]alkanoate 5a–c and dipeptides methyl-3-[2-(1,4-Dioxo-3-phenyl-3,4-dihydro-1H-phthalazin-2-yl)-acetylamino]propionylamino-alkanoates 7a–ivia the azide coupling technique. Condensation of hydrazides 6a–c with various aldehydes yielded Schiff’s base hydrazones 8a–i. Upon biological screening, results indicated that the tested compounds, especially 7c and 8b with the phenyl phthalazinone moieties, had promising cytotoxicity against the HCT-116 cells with IC50 values of 1.36 and 2.34 μM, respectively. Additionally, the promising compounds 7c and 8b exhibited poor cytotoxicity against WISH cells with much higher IC50 values, so they were safe against normal cells. Compound 8c exhibited potent anti-bacterial activity with inhibition zones of 12 and 11 mm against S. aureus and E. coli, respectively. Molecular docking results of compounds 7c and 8b revealed a good binding disposition and the ligand–receptor interactions like the co-crystallized of the VEGFR2, which may be the proposed mode of action. Finally, both compounds 7c and 8b exhibited good ADME pharmacokinetics with good drug-likeness parameters. Hence, detailed studies for the mechanism of action of such compounds are highly recommended for development of new potent anti-cancer and anti-bacterial agents.