Design, synthesis of Dioxopiperidinamide derivatives by amide coupling reaction and study of their biological activity

doi:10.21203/rs.3.rs-1655877/v1

A series of fifteen dioxopiperidinamide derivatives synthesized and their spectra chemical data were reported. An alternative amide coupling reaction has been followed by using 3-aminopiperidine-2,6-dione through amide bond functionalization between N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and Hydroxybenzotriazole (HOBt). All the designed compounds were docked in to the active sites of 2A73: Human Complement Component C3. All the docked compounds is favorable to make both hydrogen and hydrophobic interaction with amino acids located at the binding site. The most of potential compounds 4g, 4h and 7 studied for their anticancer activity on the THP-1 cancer cells line. It was also further established their Anti-inflammatory activity and Antioxidant activity with the comparable efficiency against those of clinically used drugs.

Piperidinedione Analogs attracted much attention among researchers in the recent past due to their potential bioactivty. Antineoplaston A10 is composed of 3-(phenylacetylamino)-2,6-piperidinedione, was proposed by Burzynski in 1986. Isolated from human urine and blood [1]. The potential of these analogs has been explained by their insertion between base pairs of DNA [2]. The cross-linking cellular DNA has been also reported as a strong antitumor effect, which is normally obtained from nitrogen mustards. Aniline mustard is referred to as alkylating mustard with antineoplastic activity. It forms covalent linkages with nucleophilic centers, resulting in depurination, base miscoding, strand scission, and crosslinking of DNA strands, all of which contribute to its cytotoxicity [3]. The aniline mustard containing 3-aminopiperidine-2,6-dione derivatives that are Antineoplaston A10 [4–7], Lenalidomide [8], Pomalidomide [9], Glutarimide [10], and Thalidomide [11]. These reported compounds and those structures are depicted in Fig. 1. They contain a chiral center and possess R configuration in a racemic mixture. Some of the piperidinedione derivatives with biological potent compounds are available. For example, the thalidomide has been shown proved for their antiangiogenic activities since 1990s, and its inhibition of TNF-alpha, as well as its immunomodulatory properties, therapeutic effect in multiple myeloma, were well established [12, 13]. Some of compounds in these analogs are reported to have higher potencies, like inhibition of angiogenesis or function as immunomodulatory, immunosuppressive, anticancer activity, or anti-inflammatory activities with reduced undesirable side effects [14, 15]. While designing the A1O analogs for exploring the structure-activity relations of piperidinedione and thienopyrimidine moieties in a single molecule considered owing to proven bioactivity of the individual moieties.

From the available reports, it was found that new antineoplaston A10 analogs with enhanced intravenous antitumor activity and “3-‘(4- methoxybenzoylamino)-2,6-piperidinedione’(MPD) as a microemulsion drug against MCF-7 cancer cell lines. The enhanced anticancer activity has been established through in vitro hemolytic analysis, in vivo vascular irradiation, histopathological studies, molecular docking, radiolabeling, and biodistribution studies [7].

Furthermore, the core moiety of aminoglutarimide analogs synthesized from glutamic acid [16]. By adopting the reaction conditions which was described in scheme 1. It was found that amino-glutarimides containing cyclic amide prepared from glutamicacid and formaldehyde under refluxing conditions reported to have anticonvulsant activity. In view of the above, a typical core moiety has been taken for further research in this present study.

The synthetic route of thienyl substituted derivatives as depicted in scheme 2 for the preparation of the mannich base of antineoplaston A10 analogs [17]. In this method, the first step is the reaction between 2-thiophenacetic acid and N-Hydroxy succinimide under aqueous acetonitrile solution to get thienylacetyl intermediate. Further the reaction with L-glutamine involved intramolecular cyclization to get the 3-(2-oxo-3-(thiophen-3-yl)propyl)piperidine-2,6-dione. Since this compound showed potential cytotoxicity against A459, SK-OV-3, SK-MEL-2, and XF498 cell lines compared to the simple amino piperidinedione analogs.

The design and synthesis of new phenyl acetyl substituted aminopiperidine-2,6-dione derivatives reported by Choi et al [4] as per the scheme 3. The reaction between L-glutamine and appropriate para-substituted benzoyl chlorides containing benzene under constant stirring demonstrated good yield. While analyzing their anti-cancer activity, it was found that p-methoxy-m- bis (2- chloroethyl) amine substituted by benzoyl are very active and the activity was found to be more than 1000 times better than Phenyl acetyl A10 analogs. Encouraged by these observations, this study aims to design and synthesize novel dioxopiperidinamide compounds by substituting functional groups like halogen, methoxy and methyl groups into a single molecule to investigate their cytotoxicity with promising anti-inflammatory activity.

2.1. Synthesis of dioxopiperidinamide from cinnamic acids

A series of dioxopiperidinamide compounds synthesized via amide coupling reaction. The target compounds were designed based upon molecular hybridization of potent molecules bearing 2,6-piperidinedione among the reported series. In order to understand the effect of various functionalities on their biological activity with electron-donating groups, withdrawing groups, hydrogen bond donors, and positional isomers are selected and tried. The synthetic pathway adopted is described as follows. The first step, in scheme 4 is solvent-free preparation of cinnamic acids 2a-2n with the substitution of various functional groups from the starting compounds of substituted benzaldehyde. The reaction of this substituted benzaldehyde with malonic acid is the bond expansion of the Perkin reaction. Perkin reaction facilitated to form of the corresponding cinnamic acids in the preparation of α, β unsaturated aromatic acid through an aldol condensation reaction. In this step, the anhydrous ammonium acetate without any solvents tried to complete the reaction. The yield of cinnamic acids is found to be 70–80%.

The amide coupling reaction in step 2 is routed through substituted aromatic correspond-cinnamic acids and 3-aminopiperidine-2,6-dione•HCl under Nitrogen atmosphere. The reaction is followed at room temperature, under constant stirring. The formation of the reaction product was analyzed by a thin layer chromatogram. The scheme of this reaction and Dioxopiperidincinnamamide derivatives 4a-n are represented in Scheme 5. The mechanism of this reaction between cinnamic acid and EDCl is to obtain the O-acylisourea intermediate, which is normally yielded from a very fast reaction. The second step is the involvement of the active O-acylisourea groups and HOBt to give the corresponding HOBt ester. Here the urea complex has been evaluated as a byproduct. Finally, the lone pair electron in the 3-aminopiperidine-2,6-dione abstracted with the HOBt ester to give the Dioxopiperidincinnamamide derivatives. The mechanism of reaction steps is demonstrated in Fig. 2.

2.2. Synthesis of dioxopiperidincarboxamide substituted lactam fused thienopyrimidine

To design potent substituted target compounds, a search of suitable substitutional groups in these particular analogs was conducted. Bugge et al during 2016 reported on the meta- and para amides as favorable C-6 position, contributing to an increase in activity and acting as a link for solubilizing tails. The thienopyrimidine scaffold preparation and influencing properties of anticancer activity reported [18].

Henceforth, the lactam fused thienopyrimidine in the aryl, amides, and halogen group studied to integrate the aniline hybrids that are strongly enhancing the activity rather pyrimidine and other heterocycles. Therefore, the synthesis of aniline hybrid-based reaction product with characteristic solubility effect by introducing polar functional groups and molecular planarity.

The lactam-fused thienopyrimidine synthesis and biological studies are established in our previous work [19]. From the encouraging results, the trial on the synthesis of N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10-hexahydrothieno [2',3':4,5] pyrimido [1,2-a]azepine-2-carboxamide (7) attempted from lactam fused thienopyrimidine (5) and 3-aminopiperidine-2,6-dione. The reaction's first step is ethyl ester hydrolysis followed by amide bond formation between 3-aminopiperidine-2,6-dione and lactam fused thienopyrimidine. The amide coupling reaction was carried out at room temperature, under constant stirring. The completion of the reaction was monitored by a thin layer of chromatography. The steps of this reaction are represented in Scheme 6.

The melting point of all synthesized dioxopiperidincinnamide compounds is found to correspond to the respective molecular structure and molecular weight. ¹H- and ¹³C-NMR spectroscopy data were analyzed All the target dioxopiperidincinnamide compounds possess a chiral center proton. The -CH- protons appeared in the weaker field at chemical shift value 4.62–4.67 ppm as a quartet. The presence of all phenyl rings attached methylene group in -CH₂- proton appeared in the stronger field at chemical shifts of 6.50–6.60 ppm as a doublet. Similarly, the phenyl ring aromatic –CH– proton appeared at 7.30 and 7.36 ppm as triplet spiriting.

Table 1: List of Dioxopiperidinamide derivatives

Compounds code	R¹	Melting point (°C)	Yield (%)
4a	H	260–262	47
4b	4-Cl	210–212	35
4c	4-OMe	224–226	41
4d	2,4,5-triOMe	250–252	39
4e	4-Isopropyl	200–202	40
4f	4-CH₃	218–220	45
4g	3-CH₃	190–192	45
4h	4-Br	218–220	40
4i	4-CN	258–260	38
4j	3,4-diOMe	231–233	44
4k	3,4,5-triOMePh	240–242	41
4l	2,6-diCl	230–232	30
4m	2,5-diCl	210–212	39
4n	10-chloroanthracen	282–284	25

3. Molecular Docking of dioxopiperidinamides

Molecular docking is a computational procedure that aims to predict the favored orientation of a ligand to its macromolecular target (receptor), when these are bound to each other to form a stable complex. The binding affinity (kcal/mol), distance, bonds type, and contracting amino acids for the interaction between target compounds are docked.

The receptor cavity sites are ordered in such a way that the confirmation number is in descending form and scores in increasing order. The first site is always being the most prominent site of binding normally. Site 1 of the volume of 497.25Å placed in grid spacing and angle of 0.50X.50X.50X and 90^o respectively in 3D coordinates − 60.654X -58.185Y -79.93Z. According to Ludi-based Libdock, the highest dock score was considered as the best-docked compound. In the present study, the order is found to be 7 > 4g > 4h in comparison with the standard drug Doxorubicin. All the docked compounds are favorable to both hydrogen and hydrophobic interaction with amino acids located at the binding site.

Table 2

Liplock score of all docked compounds **4f -4n** and 7.
Code	Confirmation Number	Libdock score	Atom: Atom interaction	Distance	Bond type
4g	24	102.476	B:GLU678:HN - SK31:O11	2.51944	Hydrogen Bond
			SK31:H27 - B:ASP1395:OD1	1.90146	Hydrogen Bond
			B:ARG677:HD2 - SK31:O11	2.83341	Hydrogen Bond
			SK31:H23 - B:GLN1398:OE1	2.54068	Hydrogen Bond
			B:LYS1440:NZ - SK31	4.60893	Electrostatic
			B:PRO680:HA - SK31	2.44504	Hydrophobic
			SK31:C20 - B:PRO680	5.07104	Hydrophobic
			SK31:C20 - B:ILE1363	3.39682	Hydrophobic
			SK31:C20 - B:LYS1440	3.97121	Hydrophobic
			SK31 - B:LYS1440	5.2088	Hydrophobic
4h	21	71.5393	B:ARG657:HH22 - SK32:O9	2.65252	Hydrogen Bond
			SK32:H27 - B:ASP1395:OD1	1.5832	Hydrogen Bond
			B:GLU736:OE2 - SK32	4.91258	Electrostatic
			SK32:Br20 - B:LYS1397	3.64154	Hydrophobic
			SK32 - B:LYS1397	4.34118	Hydrophobic
7			B:ARG677:HH12 - SK10:N20	2.87957	Hydrogen Bond
			SK10:H34 - B:GLU673:OE1	2.56194	Hydrogen Bond
			B:LYS670:HA - SK10:O8	3.01108	Hydrogen Bond
			SK10:S16 - B:ASP674:OD2	3.08196	Other
			B:GLU673:OE2 - SK10	3.99149	Electrostatic
			B:GLU736:OE2 - SK10	4.312	Electrostatic
			B:GLU736:OE2 - SK10	4.18202	Electrostatic
			B:LYS1397 - SK10	4.77208	Hydrophobic

Table 3 illustrated the arrived lipdock score and conformation number. It can be seen that compound 7 forms atom-atom hydrogen bonding interaction with B: ARG 677:HH12; and :N20, compound 7:H34 - B: GLU673:OE1 and K670 with a distance of 2.87 Å, 2.56 Å, and 3.011 Å respectively. Similarly, the other two ligands in compound 4f form four hydrogen bonding with GLU678, ASP1395, ARG677, GLN139, and 4g forms two favorable hydrogen bonding with ARG657 and ASP1395. Generally, the arginine and aspartic acid residues located at the binding site are favorable for hydrogen bond type whereas, Lysine (K) residue atoms are more inclined to form hydrophobic interactions to ligand. The results are corroborating these facts. Other bonding interactions are deduced with corresponding 2D and 3D interactions that are depicted in Fig. 3.

Table 3

Lipdock score of all docked compounds **4f -4n** and 7
Compound code	Libdock score	Conformation number
4f	62.78	10
4g	102.476	24
4h	71.5393	21
4i	65.43	11
4j	69.45	19
4k	70.23	48
4l	68.89	27
4m	56.78	41
4n	58.92	60
7	112.395	2
Doxorubicin	139.482	6

4.1 Cytotoxicity studies of selected Dioxopiperidinamide compounds

Based on the docking score, the selected compounds 4g, 4h and 7 have been selected for evaluation of their cytotoxic activity against THP-I cells (Human monocytic leukemia cell line) through the MTT assay method and the results are presented in Table 4. The compounds bearing alkyl group such as methyl substitution compound 4g had the best activity with the IC₅₀ value of 4.15 ug/ml and the compound 4h with the IC₅₀ value of 4.55 ug/ml. Previously Choi et al [4, 5] reported the cytotoxicity of phenyl acetyl, benzoyl, and thienyl substituted A10 analogs. From our findings, it has been established that the halogen and alkyl substitution also showed potential anticancer activity.

Table 4

Cytotoxicity studies on THP-1 cells lines
Concentration (ug/ml)	% of the dead (100-viable %)
Concentration (ug/ml)	4g	4h	7	Doxorubicin
1	51.1	41.6	45.7
5	60.3	54.45	57.7
25	72.15	66.42	68.35
125	79.68	74.15	76.2
625	83.13	79.65	80.6
IC₅₀ (ug/ml)	4.15	4.55	5.25	4.02

4.2 Anti-inflammatory activity

Target compounds were evaluated for their anti-inflammatory activity by in vitro studies using the BSA denaturation methodology and compared to the standard drug diclofenac. Table 5 summarizes the % of inhibition and concentration-dependent activity of the various Dioxopiperidincinnamamide compounds. It is seen that aromatic rings in para position with bromo substitution showed better activity than that of other substituents like cyano, methyl and methoxy groups. Anti-inflammatory activity as they are proved in the clinically approved drugs like diclofenac namely [2-(2,6-Dichloroanilino)phenyl]acetic acid with two chloro groups at the ortho position bind with Cyclooxgenases (COX) active site [20]. In view of this fact, the compounds 4f and 4h containing methyl and bromine groups also showed good % inhibition of 94.41% and 95.32% at 500 µg/ml (Table 5) while Diclofenac efficiency is 93.31%.

Table 5

Anti-inflammatory activity of Dioxopiperidincinnamamides
Conc µg/ml	Compounds code								STD
Conc µg/ml	4a	4b	4c	4d	4e	4f	4h	4j	STD
10	11.62	10.98	9.45	13.21	12.98	16.27	18.62	8.73	16.23
50	15.55	13.45	12.28	20.62	17.69	23.67	26.71	11.11	24.32
100	46.24	44.75	42.25	52.39	49.45	55.89	57.44	40.91	56.25
250	68.72	65.33	63.35	73.38	70.62	76.01	77.89	61.14	76.66
500	85.73	83.17	80.42	90.47	88.33	94.41	95.32	77.44	93.31

4.3. Antioxidant activity

The Antioxidant activity was examined by performing % inhibition on the Phosphomolybdenum method. The antioxidant reagent was treated with various concentrations (10–500 µg/ml) of derivatives after which the percentage of inhibition was calculated. The results of all tested compounds presented in Table 6 and the efficiency has compared with Ascorbic acid as standard compound. Ascorbic acid having a phenol group in the crucial cite enhance the antioxidative action by directly contributing in breaking of antioxidants [21]. The present study the compounds with substitutions groups of alkyl and halogen groups tested and showed least Antioxidant activity.

Table 6

Antioxidant activity of Dioxopiperidincinnamamides
Conc µg/ml	Compounds								STD
Conc µg/ml	4a	4b	4c	4d	4e	4f	4h	4j	STD
10	5	4.1	5.71	6.98	7.1	3.5	2.91	2.22	14.87
50	10.33	8.66	10.73	11.27	11.42	6.92	5.73	5.02	29.42
100	14.79	12.37	16.21	17.14	17.66	10.44	9.79	9.1	48.74
250	22.96	20.81	23.35	24.98	25.49	17.21	16.36	15.42	75.32
500	30.91	28.66	32.44	35.02	35.32	24.66	23.31	22.19	98.9

4.4. Structure Activity Relationship

While testing the anticancer activity in vitro using MTT Assay method, the methyl substituted compound 4g showed better than bulky group substitution compound 7. The Antioxidant activity and anti-inflammatory activity in vitro using the BSA denaturation model, it is observed phenyl ring fused compounds methyl and bromo group substitution shows better activity than that other substituted compounds. It implies that methoxy group substitution in compounds 4j and 4c hampers the anti-inflammatory activity. It is well known that functional groups like chloro, bromo and methyl groups functionally modify the Dioxopiperidincinnamamides moiety, thereby exhibiting potential anti-inflammatory activity and Antioxidant activity. However, the interactions are purely based on the position of substituents and orientation of the aryl groups. Therefore, we are suggested a SAR combination approach to consider the positioning to form intramolecular hydrogen bonding or strong hydrogen acceptor compounds showing moderate activity.

5.1. The general synthetic procedure for preparation of substituted cinnamic acid (2a-2n) derivatives

A mixture of appropriate benzaldehyde (40 mmol), malonic acid (4.18g, 40 mmol), and ammonium acetate (3.08g, 40 mmol) was mixed in an RB flask and heated 110–120°C on oil-bath reflux without a solvent for four hours. The reaction started very quickly: in two minutes the whole reaction mixture fused to a homogeneous liquid. In another five minutes, a cream-colored solid appeared, and the whole reaction mixture had become perfectly solid formed before the heating was stopped. It was allowed to cool, treated with crushed ice and the reaction mixture was stirred for 1 hour. The colored precipitate was obtained. The precipitate was filtered and recrystallized from methanol to afford pure cinnamic acid derivatives 2a-n.

5.2. The general synthetic procedure for preparation of Dioxopiperidincinnamamide derivatives (4a-4n)

A solution of EDC•HCl (1.164 g, 0.006’mol) and HOBt (0.820 g, 0.006’mol) in CH₂Cl₂ (10’mL) was added dropwise to a stirred suspension of cinnamic acids (6.07 mmol) and 3-aminopiperidine-2,6-dione•HCl (1.0g, 6.07 mmol) in CH₂Cl₂ (15 mL) at 0°C under Nitrogen atmosphere. Triethylamine (1.70 mL, 12.14 mmol) was added dropwise to the mixture, which was then stirred at 25°C overnight. The reaction was completed confirmed by TLC. The reaction mass was quenched with ethyl acetate and concentrated with an organic layer followed by column chromatography purification using dichloromethane and methanol (9:2) to obtain pure compounds 4a-4n.

5.3. General procedure for the preparation of 3-methyl-4-oxo-4,6,7,8,9,10-hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxylic acid (6)

Compounds 5 was solubilized in 8% methanol/ 4% water, and 2ml of 15% v/v aqueous sodium hydroxide solution was added. Stirring was continued for 16 hrs at room temperature, then CHCl₃ was added. The 1N HCl was added to neutralize the aqueous layer, further stirred for 30 min, the product was filtered by vacuum and washed with DM water, suck dried for 1hr and recrystallized from Dichloromethane and methanol to give compounds 6 in good yield.

5.4. The general synthetic procedure for preparation of N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10 hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxamide (7)

A solution of EDC•HCl (1.164 g, 6.07 mmol) and HOBt (0.820 g, 6.07 mmol) in CH₂Cl₂ (10 mL) was added dropwise to a stirred suspension of compounds 5 (6.07 mmol) and 3-aminopiperidine-2,6-dione•HCl (1.0g, 6.07 mmol) in CH₂Cl₂ (15 mL) at 0°C under Nitrogen atmosphere. Triethylamine (1.70 mL, 12.14 mmol) was added dropwise to the mixture, which was then stirred at RT overnight. The purification technique was followed as preparation of section 5.2 derivatives

5.5. ¹H and ¹³C NMR data and their analysis

N-(2,6-dioxopiperidin-3-yl)cinnamamide (4a): ¹H NMR (500 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.50 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 15.8 Hz, 2H), 7.43 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.4 Hz, 2H), 6.71 (d, J = 15.8 Hz, 1H), 4.71 ( q, J = 8.4 Hz, 1H), 2.82–2.75 (m, 2H), 2.02–1.97 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.74, 172.51, 165.83, 140.42, 134.92, 130.31, 129.49, 128.15, 121.56, 49.71, 31.26, 24.58.

(E)-3-(4-chlorophenyl)-N-(2,6-dioxopiperidin-3-yl)acrylamide (4b)

¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 8.76 (d, J = 2.2 Hz, 1H), 8.43 (d, J = 4.8 Hz, 1H), 8.21 (d, J = 5.6 Hz, 2H), 7.58 (d, J = 10.6 Hz, 2H), 7.03 (d, J = 9.0 Hz, 1H), 4.77 (q, J = 9.6 Hz, 1H), 2.82–2.65 (m, 2H), 2.05–1.92 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.30, 172.60, 165.50, 138.08, 133.09, 129.82, 129.72, 128.90, 128.78, 50.00, 31.44, 24.60.

(E)-N-(2,6-dioxopiperidin- 3-yl)-3-(4-methoxyphenyl)acrylamide (4c)

¹H NMR (500 MHz, DMSO) δ 10.85 (s, 1H), 8.37 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.43 (d, J = 15.8 Hz, 2H), 6.99 (t, J = 8.7 Hz, 2H), 6.56 (d, J = 15.8 Hz, 1H), 4.70 (q, J = 2.7 Hz, 1H), 3.80 (s, 3H), 2.86–2.69 (m, 2H), 2.00–1.94 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.47, 172.77, 165.75, 160.92, 139.67, 129.70, 127.77, 119.57, 114.89, 55.73, 49.70, 31.15, 24.90.

(E)-N-(2,6-dioxopiperidin- 3-yl)-3-(3,4,5-trimethoxyphenyl)acrylamide (4d)

¹H NMR (500 MHz, DMSO) δ 10.88 (s, 1H), 8.39 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 15.7 Hz, 1H), 6.94 (s, 2H), 6.68 (d, J = 15.7 Hz, 1H), 4.71 (q, J = 12.0, 6.0 Hz, 1H), 3.36 (s, 9H), 2.83–2.72 (m, 2H), 2.11–1.91 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.43, 172.73, 165.48, 153.55, 140.01, 139.19, 130.88, 121.55, 105.49, 60.55, 56.33, 49.72, 31.38, 24.89.

(E)-N-(2,6-dioxopiperidin- 3-yl)-3-(4-isopropylphenyl)acrylamide (4e)

¹H NMR (500 MHz, DMSO) δ 10.88 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 15.8 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 13.8 Hz, 1H), 4.71 (q, J = 7.9 Hz, 1H), 2.94–2.88 (m, 1H), 2.81–2.76 (m, 2H), 2.04–1.94 (m, 2H), 1.21 (d, J = 6.8 Hz, 6H). ¹³C NMR (126 MHz, DMSO) δ 173.42, 172.75, 165.53, 150.66, 143.29, 139.84, 132.90, 126.71, 121.17, 49.72, 33.77, 31.41, 24.90, 24.13.

(E)-N-(2,6-dioxopiperidin- 3-yl)-3-p-tolylacrylamide (4f)

¹H NMR (500 MHz, DMSO) δ 10.94 (s, 1H), 8.63 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 15.8 Hz, 2H), 7.32 (d, J = 7.5 Hz, 2H), 6.70 (d, J = 15.8 Hz, 1H), 4.74 (q, J = 8.0 Hz, 1H), 2.87–2.78 (m, 2H), 2.40 (s, 3H), 2.36–1.77 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.67, 172.54, 165.92, 140.35, 140.20, 132.19, 130.08, 128.15, 120.52, 49.64, 31.26, 24.61, 21.37.

(E)-N-(2,6-dioxopiperidin- 3-yl)-3-(m-tolyl)acrylamide (4g)

¹H NMR (500 MHz, DMSO) δ 10.87 (s, 1H), 8.62 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 6.5 Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.26 (d, J = 7.4 Hz, 1H), 6.71 (d, J = 8.8 Hz, 1H), 4.71 (q, J = 8.0 Hz, 1H), 2.85–2.74 (m, 2H), 2.37 (s, 3H), 2.11–1.99 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.65, 172.51, 165.81, 140.48, 138.74, 134.91, 130.98, 129.37, 128.69, 125.29, 121.43, 49.67, 31.26, 24.61, 21.28.

(E)-3-(4-bromophenyl)-N-(2,6-dioxopiperidin-3-yl)acrylamide (4h)

¹H NMR (500 MHz, DMSO) δ 10.78 (s, 1H), 8.60 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 6.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 6.68 (d, J = 8.0 Hz, 2H), 4.65 (d, J = 8.7 Hz, 1H), 2.79–2.68 (m, 2H), 2.05–1.95 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.63, 172.44, 165.53, 139.08, 134.28, 132.39, 130.12, 123.41, 122.51, 49.70, 31.25, 24.56.

(E)-3-(4-cyanophenyl)-N-(2,6-dioxopiperidin-3-yl)acrylamide (4i)

¹H NMR (500 MHz, DMSO) δ 10.87 (s, 1H), 8.71 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 15.8 Hz, 2H), 6.80 (d, J = 15.8 Hz, 1H), 4.65 (q, J = 8.2 Hz, 1H), 2.80–2.62 (m, 2H), 2.12–1.84 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.63, 172.37, 165.14, 139.65, 138.49, 133.26, 128.87, 125.11, 119.15, 112.02, 49.76, 31.24, 24.50.

(E)-3-(3,4-dimethoxyphenyl)- N-(2,6-dioxopiperidin-3-yl)acrylamide (4j)

¹H NMR (500 MHz, DMSO) δ 10.87 (s, 1H), 8.71 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 15.8 Hz, 1H), 6.80 (d, J = 15.8 Hz, 1H), 4.67 (d, J = 8.5 Hz, 1H), 2.86–2.58 (m, 2H), 2.15–1.79 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.65, 172.58, 166.08, 150.71, 149.28, 140.43, 127.81, 122.26, 119.33, 112.10, 110.34, 79.35, 55.88, 49.64, 31.26, 24.67.

(E)-3-(3,4-dichlorophenyl)- N-(2,6-dioxopiperidin-3-yl)acrylamide (4k)

¹H NMR (500 MHz, DMSO) δ 10.86 (s, 1H), 8.63 (d, J = 8.2 Hz, 1H), 7.87 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.42 (s, 1H), 6.73 (d, J = 15.8 Hz, 1H), 4.66 (q, J = 7.1, 4.9 Hz, 1H), 2.87–2.63 (m, 2H), 2.10–1.82 (m, 2H). ¹³C NMR (126 MHz, CDCl3) δ 178.39, 177.15, 169.99, 142.53, 140.62, 137.18, 136.96, 134.72, 134.60, 132.92, 128.61, 54.48, 29.26, 19.21.

(E)-3-(2,6-dichlorophenyl)-N-(2,6-dioxopiperidin-3-yl)acrylamide (4l)

¹H NMR (500 MHz, DMSO) δ 10.93 (s, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.5Hz, 1H), 7.60 (t, J = 4.7 Hz, 1H), 7.57 (d, J = 6.0 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H), 6.81 (d, J = 6.1 Hz, 1H), 4.73 (q, J = 6.8 Hz, 1H), 2.83–2.78 (m, 2H), 2.11–2.04 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.61, 172.31, 164.72, 134.26, 133.78, 132.18, 131.11, 129.93, 129.66, 129.55, 120.16, 49.76, 31.22, 24.51.

(E)-3-(2,5-dichlorophenyl)- N-(2,6-dioxopiperidin-3-yl)acrylamide (4m)

¹H NMR (500 MHz, DMSO) δ 10.87 (s, 1H), 8.90 (d, J = 6.4 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 6.77 (d, J = 15.7 Hz, 1H), 6.57 (s, 1H), 4.65 (q, J = 8.8 Hz, 1H), 2.78–2.60 (m, 2H), 1.97–1.74 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 173.61, 167.99, 143.19, 129.65, 127.87, 127.61, 126.07, 124.20, 120.15, 110.11, 106.66, 49.81, 31.22, 24.46.

(E)-3-(10-chloroanthracen- 9-yl)-N-(2,6-dioxopiperidin-3-yl)acrylamide (4n)

¹H NMR (500 MHz, DMSO) δ 10.92 (s, 1H), 8.84 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.30 (t, J = 12.8 Hz, 2H), 8.20 (t, J = 8.7 Hz, 1H), 7.71 (d, J = 7.9 Hz, 2H), 7.64 (d, J = 6.9 Hz, 2H), 6.53 (d, J = 16.1 Hz, 1H), 4.77 (q, J = 5.7 Hz, 1H), 2.82–2.76 (m, 2H), 2.10–2.06 (m,2H). ¹³C NMR (126 MHz, DMSO) δ 173.66, 173.11, 164.96, 136.48, 136.35, 131.41, 130.69, 129.49, 128.61, 127.97, 126.64, 126.10, 125.93, 124.96, 49.86, 31.28, 24.62.

N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10- hexahydrothieno 2',3':4,5] pyrimido [1,2-a]azepine-2-carboxamide (7): ¹H NMR (500 MHz, DMSO) δ 10.90 (s, 1H), 8.45 (d, J = 8.3 Hz, 1H), 4.74 (q, J = 7.9, 5.1 Hz, 1H), 4.31 (t, J = 4.2 Hz, 2H), 3.04 (d, J = 3.1 Hz, 2H), 2.74 (s, 3H), 2.20–2.10 (m, 2H), 2.01–1.95 (m, 2H), 176 − 1.67 (m, 6H). 13C NMR (126 MHz, DMSO) δ 173.44, 172.41, 163.27, 163.20, 162.35, 158.74, 137.41, 127.19, 120.72, 50.27, 41.98, 37.07, 31.43, 29.20, 27.28, 24.84, 24.48, 15.45.

A series of fifteen dioxopiperidinamide derivatives synthesized by an amide coupling reaction and docked to ascertain their effective binding to relate cytotoxicity. The results are encouraging for further drug design. The concentration responsive capacity of the compounds tested on the THP-1 cancer cells line. Dioxopiperidincinnamide 4g and 4h derivatives are potent anticancer activity against THP-1 cells line with IC₅₀ value is 4.15 and 4.55 ug/ml respectively, while compound 7 showed slightly lower efficacy. However, all these compounds showed more or less equal to standard Doxorubicin drug having IC₅₀: 4.02 ug/ml. Compounds 7, thienopyrimidine substituted derivative showed anticancer activity against THP-I cells with IC₅₀ value 5.25 ug/ml. The structure-activity relation revealed that lack of chirality and the presence of bulky substituents in a few of the compounds are found to be the cause for lower potency. The Anti-inflammatory activity against the BSA denaturation technique revealed that 4-Bromo substituted dioxopiperidincinnamide (4h) showed moderate inhibitory activity compared to that of other synthesized Dioxopiperidin cinnamamide derivatives. 4-Bromo substituted dioxopiperidincinnamide (4h) regarded as a lead compound from the study results.

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Schemes 1 To 6 are available in the Supplementary Files section

Scheme1.jpg
Synthesis of aminoglutarimides
Scheme2.jpg
Synthesis of (3-(thiophen-3-yl)propyl)piperidine-2,6-dione
Scheme3.jpg
Synthesis of a phenyl ring of A10 analogs
Scheme4.jpg
General synthetic scheme of Cinnamic acid derivatives 2a-2n
Scheme5.jpg
General synthetic scheme of Dioxopiperidincinnamamide derivatives 4a-4n
Scheme6.jpg
Synthesis of N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10-hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxamide (7)

Design, synthesis of Dioxopiperidinamide derivatives by amide coupling reaction and study of their biological activity

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results And Discussion

2.1. Synthesis of dioxopiperidinamide from cinnamic acids

2.2. Synthesis of dioxopiperidincarboxamide substituted lactam fused thienopyrimidine

3. Molecular Docking Of Dioxopiperidinamides

4. Biological Activity

4.1 Cytotoxicity studies of selected Dioxopiperidinamide compounds

4.2 Anti-inflammatory activity

4.3. Antioxidant activity

4.4. Structure Activity Relationship

5. Experimental Section

5.1. The general synthetic procedure for preparation of substituted cinnamic acid (2a-2n) derivatives

5.2. The general synthetic procedure for preparation of Dioxopiperidincinnamamide derivatives (4a-4n)

5.3. General procedure for the preparation of 3-methyl-4-oxo-4,6,7,8,9,10-hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxylic acid (6)

5.4. The general synthetic procedure for preparation of N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10 hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxamide (7)

5.5. ¹H and ¹³C NMR data and their analysis

6. Conclusion

References

Schemes

Supplementary Files

Status:

Version 1

Design, synthesis of Dioxopiperidinamide derivatives by amide coupling reaction and study of their biological activity

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results And Discussion

2.1. Synthesis of dioxopiperidinamide from cinnamic acids

2.2. Synthesis of dioxopiperidincarboxamide substituted lactam fused thienopyrimidine

3. Molecular Docking Of Dioxopiperidinamides

4. Biological Activity

4.1 Cytotoxicity studies of selected Dioxopiperidinamide compounds

4.2 Anti-inflammatory activity

4.3. Antioxidant activity

4.4. Structure Activity Relationship

5. Experimental Section

5.1. The general synthetic procedure for preparation of substituted cinnamic acid (2a-2n) derivatives

5.2. The general synthetic procedure for preparation of Dioxopiperidincinnamamide derivatives (4a-4n)

5.3. General procedure for the preparation of 3-methyl-4-oxo-4,6,7,8,9,10-hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxylic acid (6)

5.4. The general synthetic procedure for preparation of N-(2,6-dioxopiperidin-3-yl)-3-methyl-4-oxo-4,6,7,8,9,10 hexahydrothieno[2',3':4,5]pyrimido[1,2-a]azepine-2-carboxamide (7)

5.5. 1H and 13C NMR data and their analysis

6. Conclusion

References

Schemes

Supplementary Files

Status:

Version 1

5.5. ¹H and ¹³C NMR data and their analysis