Synthesis and in vitro anticancer activities of substituted N-(4′-nitrophenyl)-l-prolinamides

Abstract

Prolinamides are present in secondary metabolites and have wide-ranging biological properties as well as antimicrobial and cytotoxic activities. N-(4′-substituted phenyl)-l-prolinamides 4a–4w were synthesized in two steps, starting from the condensation of p-fluoronitrobenzene 1a–1b with l-proline 2a–2b, under aqueous–alcoholic basic conditions to afford N-aryl-l-prolines 3a–3c, which underwent amidation via a two-stage, one-pot reaction involving SOCl₂ and amines, to furnish l-prolinamides in 20–80% yield. The cytotoxicities of 4a–4w against four human carcinoma cell lines (SGC7901, HCT-116, HepG2 and A549) were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; with good tumour inhibitory activities (79.50 ± 1.24%–50.04 ± 1.45%) against HepG2. 4a exhibited the best anti-tumour activity against A549 with percentage cell inhibition of 95.41 ± 0.67% at 100 µM. Likewise, 4s (70.13 ± 3.41%) and 4u (83.36 ± 1.70%) displayed stronger antineoplastic potencies against A549 than the standard, 5-fluorouracil (64.29 ± 2.09%), whereas 4a (93.33 ± 1.36%) and 4u (81.29 ± 2.32%) outperformed the reference (81.20 ± 0.08%) against HCT-116. SGC7901 showed lower percentage cell viabilities with 4u (8.02 ± 1.54%) and 4w (27.27 ± 2.38%). These results underscore the antiproliferative efficacies of l-prolinamides while exposing 4a and 4u as promising broad-spectrum anti-cancer agents. Structure-activity relationship studies are discussed.

1. Introduction

Cancer is the uncontrolled growth and spread of cells. It is a non-communicable disease but remains the second leading cause of mortality worldwide, with over 9.6 million estimated deaths and 18.1 million new cases annually [1,2]. The identification and syntheses of chemical anti-cancer agents with wide-ranging inhibitory activities against tumour cells have continued to stimulate research across disciplines [3–5]. Though several therapy protocols have been applied in the treatment of the different types of cancer, their non-selectivity towards target tumour cells and cytotoxicity to normal cells as well as multidrug resistance, non-bioavailability and severe side effects are major limitations [6].

Peptides are typically accessed in the development of new classes of therapeutic agents because of their high activity, low immunogenicity, good biocompatibility and amenability to synthetic modifications [4] but naturally occurring peptides are often unsuitable therapeutics because of such limitations as poor chemical and physical stabilities, as well as short circulating plasma half-lives. The application of synthetic peptides has also been severely restricted by poor membrane permeability, high clearance, low systemic stability and negligible activity when administered orally [7]. However, various strategies, such as the inclusion of non-natural amino acids and their conjugation with sugars, lipids and proteins as well as the use of polyvalent peptide synthesis, have been adopted to overcome these drawbacks [8].

The N-functionalization of amino acids with aryl compounds has great use in organic syntheses as well as drug discovery and pharmaceutics [9,10]. N-aryl amino acids are, therefore, employed as inexpensive chiral building blocks and are crucial motifs in many systems of physiological importance [11]. N-arylated amino acid derivatives can also be incorporated into peptides and proteins to propel the development of new methods for the study of protein structures and functions, among others [7]. Pertinently, N-arylation reactions have been exploited in the introduction of diversity into bioactive molecules and synthesis of anti-cancer agents with improved potencies [10].

Prolinamides are carboxamides of the cyclic amino acid, proline and, therefore, possess the electronic and spatial properties of the amide bonds inherent in peptides as well as their bent secondary structures. Prolinamides find uses as reverse-turn mimetics [12], ligands and asymmetric organocatalysts [13]. Furthermore, the prolinamide moiety abounds in several molecules of biological and therapeutic importance [7], and some of these compounds have been reported to possess inhibitory activities against tumour cells [14–16] (figure 1).

Figure 1.

Some prolinamide-based anti-cancer compounds (Abarelix [7], Phosmidosine [14] and Pro-4 [17]).

Prolinamide derivatives can preferentially target i-motifs and G-quadruplexes, which are dynamic nucleic acid secondary structures, believed to play key roles in gene expression [18]. The selective recognition of the c-MYC G-quadruplex DNA by compounds with the prolinamide motif has been recently demonstrated [17]. The c-MYC oncogene is associated with cell growth, proliferation and a range of malignant tumours. Therefore, the use of prolinamides to target the promoter region of oncogenes, such as c-MYC and BCL-2, is a promising route towards developing effective cancer therapies. Additionally, the N-arylation of prolinamides, if synergistic, should bode well for the resulting substrates' antineoplastic potencies.

Presented herein, are the synthesis and characterization of a series of N-(4′-nitrophenyl)-l-prolinamides via a simple two-step reaction, starting from easily available and inexpensive p-halogenonitrobenzenes and l-proline. Impelled by the literature precedents [7,14–18] of prolinamide-containing anti-tumour agents; dating back to actinomycin [19], four of the most commonly occurring types of human cancers [20] (colon, liver, lung and gastric) were also selected for in vitro assays. Consequently, the anti-cancer properties of the synthesized l-prolinamides were evaluated against human colon (HCT-116), liver (HepG2), lung (A549) and gastric (SGC7901) carcinoma cell lines.

2. Results and discussion

2.1. Chemistry

The requisite N-(4′-nitrophenyl)-l-prolinamides 4a–w were accessed via a two-step reaction as shown in scheme 1. The first step involved a base-catalysed condensation reaction, using potassium carbonate in refluxing ethanol–water (1 : 1) solution, as earlier reported [21,22], to furnish N-(4′-nitrophenyl)-l-prolines 3a–c in 70–90% yield. It was found that refluxing the reaction in ethanol–water (1 : 1) solution was marginally better than refluxing in ethanol alone. p-Fluoronitrobenzene 1a was the halogenobenzene of choice because fluorine is a good leaving group and typically affords better-yielding products in reactions as those that proceed via a nucleophilic aromatic substitution (S_NAr) mechanism [23,24].

Scheme 1.

Synthetic route to N-(4′-nitrophenyl)-l-prolinamides.

The amidation of the N-(4′-nitrophenyl)-l-proline adducts 3a–3c with appropriate amines produced the target N-(4′-nitrophenyl)-l-prolinamides 4a–4w in a two-stage, one-pot reaction [25,26]. Different amines were employed in the amidation reaction with a view to obtaining potent anti-tumour agents with broad-spectrum activities and evaluating structure–activity relationships on their cytotoxicities. These included using primary, secondary, linear or branched amines as well as sulfonamides (table 1).

Table 1.

Synthesis of substituted N-(4′-nitrophenyl)-l-prolinamides (4a–w).

The first stage of the one-pot amidation reaction involved the activation of the acid adducts 3a–3c, into N-(4′-nitrophenyl)pyrrolidine-2-carboxylic acid chlorides 5, via a modified procedure [25], with thionyl chloride (SOCl₂). It is noteworthy that the complete removal of excess SOCl₂ in situ from the reaction medium (without exposure to moisture) was crucial to the success of the chlorination reaction. We also found that using stoichiometric equivalents of SOCl₂ resulted in incomplete reactions. The addition of N,N-dimethylformamide (one drop) appeared to have a catalytic effect on the formation of the acid chlorides; improving yield and reaction time.

It is noteworthy that all the synthesized l-prolinamides are solid, with melting points ranging from 74–75°C (4a) to 252–253°C (4e); with the exception of 4l, which is a viscous oil. The prevalence of the l-prolinamides in the solid-state alludes to the presence of intra- and intermolecular hydrogen bonding and π–π-stacking.

Considering the infrared (IR) spectra of the acid adducts 3a–3c and prolinamides 4a–4w, the disappearance of the broad acid O–H stretching bands, observed between 3400 and 3200 cm^–1 in the IR spectra of 3a–3c, and the downward shift of the carbonyl (C=O) stretching frequencies, from 1734–1714 cm^–1 in 3a–3c, to 1687–1637 cm^–1 in the IR spectra of 4a–4w were both indicative of a successful transformation. As was the appearance of the N–H stretching bands between 3318 cm^–1 and 3248 cm^–1 in the IR spectra of the secondary amides. The ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectroscopic data as well as mass spectrometry analysis are also in agreement with the assigned structures. Notably, the presence of rotamers (1 : 1) was evident in the ¹H- and ¹³C-NMR spectra of 4a, 4e, 4j, 4o and 4w. The target N-(4′-nitrophenyl)-l-prolinamides 4a–4w were obtained in 20–80% yield, after purification by column chromatography on silica gel, with n-hexane and ethyl acetate (2 : 1) as mobile phase.

2.2. Biology

The cytotoxicity analyses of l-prolinamides 4a–4w were conducted in vitro with four different human cancer cell lines: gastric carcinoma (SGC7901), colon carcinoma (HCT-116), liver carcinoma (HepG2) and lung carcinoma (A549), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [27,28]. The antiproliferative efficacy data were obtained from the cell viability assay calculations of the four carcinoma cell lines after their treatment with the l-prolinamide substrates. Cellular viability assays are generally used to quantify the number of healthy cells in a sample, while cell viability represents the number of healthy cells present in a given population [29]. The percentage cell viability, on the other hand, is the ratio of the healthy cells to the total cell population expressed as a percentage. Herein, three different prolinamide concentrations (1 µM, 10 µM and 100 µM) were incubated with each of the cell lines (cf. table 2) and 5-fluorouracil, which is an antimetabolite with antineoplastic activities used to treat multiple solid tumours, was employed as the positive control.

Table 2.

Percentage cell viability (MTT assay) of l-prolinamides against four human carcinoma cell lines. (SGC7901, human gastric carcinoma cell line; HCT-116, human colon carcinoma cell line; HepG2, human liver carcinoma cell line; A549, human lung carcinoma cell line; 5-FU, 5-fluorouracil (positive control); n.d., not determined.)

l-prolinamide			carcinoma cell lines
			gastric			colon			liver			lung
			SGC7901			HCT-116			HepG2			A549
			1 µM	10 µM (%)	100 µM	1 µM	10 µM (%)	100 µM	1 µM	10 µM (%)	100 µM	1 µM	10 µM (%)	100 µM
1.	4a		91.98 ± 9.12	87.15 ± 5.50	95.35 ± 2.26	95.11 ± 4.04	86.87 ± 5.90	6.67 ± 1.36	95.41 ± 0.71	96.49 ± 1.27	24.95 ± 4.09	95.07 ± 2.65	93.35 ± 1.30	4.59 ± 0.67
2.	4b		111.41 ± 20.13	111.98 ± 21.71	115.12 ± 17.74	93.21 ± 6.34	87.534.06	61.17 ± ±4.16	95.46 ± 4.09	88.58 ± 3.60	77.89 ± 6.34	90.41 ± 1.90	85.23 ± 2.89	81.52 ± 4.12
3.	4c		121.46 ± 12.83	112.23 ± 11.01	97.08 ± 9.08	95.90 ± 6.37	95.80 ± 4.64	91.37 ± 0.95	88.19 ± 1.65	89.42 ± 2.54	87.36 ± 3.72	63.01 ± 6.75	51.84 ± 4.33	52.11 ± 3.28
4.	4d		109.60 ± 5.81	101.99 ± 17.20	100.48 ± 15.88	96.58 ± 7.44	86.47 ± 5.47	81.47 ± 5.75	94.10 ± 0.98	91.10 ± 2.57	94.44 ± 3.38	85.14 ± 2.29	77.15 ± 5.95	64.43 ± 5.47
5.	4e		86.70 ± 5.20	84.72 ± 4.16	87.10 ± 9.56	88.42 ± 7.89	81.12 ± 5.51	67.79 ± 4.68	88.95 ± 3.60	77.02 ± 6.91	20.50 ± 1.24	54.11 ± 4.96	52.85 ± 4.13	42.22 ± 4.65
6.	4f		87.01 ± 6.85	88.25 ± 6.02	94.48 ± 13.60	94.49 ± 6.77	96.95 ± 3.69	86.99 ± 6.33	87.53 ± 1.04	92.92 ± 4.76	102.19 ± 3.43	122.08 ± 8.43	110.09 ± 3.43	102.53 ± 2.20
7.	4g		89.41 ± 2.56	93.51 ± 3.62	90.72 ± 14.06	91.96 ± 9.11	95.97 ± 6.02	76.76 ± 7.02	89.00 ± 1.32	89.22 ± 1.29	84.12 ± 0.90	95.68 ± 8.49	95.24 ± 6.32	84.85 ± 4.19
8.	4h		95.71 ± 4.25	86.12 ± 4.63	71.05 ± 0.61	95.63 ± 6.37	104.93 ± 4.64	96.64 ± 0.95	79.90 ± 2.26	89.20 ± 2.05	94.03 ± 2.77	70.15 ± 9.28	72.09 ± 3.93	91.46 ± 4.20
9.	4i		90.08 ± 17.70	89.37 ± 13.72	77.68 ± 4.87	96.39 ± 6.39	92.24 ± 6.15	93.13 ± 7.95	82.36 ± 5.24	87.61 ± 4.24	85.84 ± 1.10	114.10 ± 6.56	97.28 ± 10.58	79.81 ± 2.95
10.	4j		81.51 ± 2.90	83.58 ± 1.95	68.41 ± 4.88	101.49 ± 3.59	101.04 ± 4.66	108.82 ± 2.47	82.38 ± 4.23	90.20 ± 4.67	94.13 ± 6.04	81.19 ± 0.83	72.08 ± 6.14	72.07 ± 2.65
11.	4k		83.18 ± 10.33	77.91 ± 5.00	68.38 ± 11.07	103.04 ± 5.72	93.97 ± 8.24	70.33 ± 13.79	92.60 ± 2.16	102.06 ± 3.45	35.81 ± 4.22	120.22 ± 5.79	141.96 ± 10.00	150.46 ± 6.90
12.	4l		n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
13.	4m		89.14 ± 2.30	93.90 ± 7.98	90.95 ± 2.14	87.89 ± 4.77	84.35 ± 1.62	81.84 ± 3.69	94.37 ± 1.95	83.17 ± 5.41	68.24 ± 7.67	167.13 ± 0.81	161.42 ± 10.99	135.98 ± 6.08
14	4n		92.73 ± 3.19	85.55 ± 4.37	89.71 ± 10.43	101.90 ± 6.83	110.42 ± 3.53	79.94 ± 3.74	117.36 ± 2.96	84.46 ± 8.45	60.78 ± 4.50	99.76 ± 12.48	105.15 ± 11.46	68.48 ± 3.32
15.	4o		85.49 ± 11.10	83.39 ± 9.62	73.41 ± 4.85	107.06 ± 3.58	100.34 ± 8.79	72.92 ± 7.73	91.59 ± 4.76	84.62 ± 3.99	38.20 ± 6.03	111.35 ± 17.37	101.55 ± 11.08	55.56 ± 9.08
16.	4p		94.35 ± 7.09	83.34 ± 6.22	96.06 ± 2.34	92.89 ± 3.16	109.73 ± 4.504	72.54 ± 5.35	91.09 ± 9.17	94.76 ± 4.14	90.43 ± 8.30	117.56 ± 17.21	127.63 ± 18.70	91.32 ± 2.86
17.	4q		102.17 ± 4.77	95.09 ± 6.27	32.98 ± 1.03	94.55 ± 5.94	100.79 ± 8.68	50.37 ± 2.34	82.81 ± 4.56	84.34 ± 8.14	49.96 ± 1.45	122.48 ± 23.81	124.75 ± 4.92	62.09 ± 0.98
18.	4r		78.45 ± 3.54	82.10 ± 3.88	85.08 ± 2.17	94.86 ± 2.68	99.20 ± 3.57	68.99 ± 1.89	91.59 ± 4.76	84.62 ± 3.99	38.20 ± 6.03	87.77 ± 13.65	89.82 ± 11.28	54.59 ± 7.13
19.	4s		93.71 ± 6.65	100.02 ± 1.64	39.02 ± 9.12	102.68 ± 10.84	113.41 ± 6.07	72.44 ± 0.388	91.29 ± 6.29	77.23 ± 1.96	41.82 ± 2.96	82.48 ± 4.2	91.14 ± 11.46	29.87 ± 3.41
20.	4t		65.87 ± 4.25	93.87 ± 4.17	87.03 ± 4.83	106.00 ± 0.81	103.46 ± 5.29	102.87 ± 7.41	93.54 ± 3.84	98.32 ± 2.09	101.52 ± 3.42	104.43 ± 6.09	121.39 ± 6.08	70.29 ± 8.39
21.	4u		96.92 ± 1.46	85.39 ± 1.23	8.02 ± 1.54	103.74 ± 9.51	88.03 ± 3.17	18.71 ± 2.32	91.20 ± 2.47	71.81 ± 2.32	28.04 ± 0.99	104.02 ± 3.18	50.13 ± 6.42	16.64 ± 1.70
22.	4v		92.92 ± 4.26	97.98 ± 6.59	57.29 ± 5.64	99.38 ± 2.28	107.71 ± 10.25	104.73 ± 7.67	82.72 ± 0.91	89.29 ± 1.69	87.80 ± 3.02	60.38 ± 5.18	65.96 ± 4.84	75.90 ± 6.26
23.	4w		80.28 ± 1.46	74.71 ± 11.69	27.27 ± 2.38	102.04 ± 1.91	90.11 ± 7.77	38.90 ± 2.70	94.36 ± 0.31	89.72 ± 2.26	48.91 ± 1.17	103.13 ± 2.62	97.43 ± 9.23	45.60 ± 3.65
24.	5-FU		81.77 ± 4.89	57.94 ± 7.11	31.28 ± 5.83	57.38 ± 3.18	44.64 ± 4.80	18.80 ± 0.08	88.18 ± 1.76	25.62 ± 3.29	9.03 ± 1.29	67.80 ± 1.41	42.04 ± 2.36	35.71 ± 2.09

Table 2 highlights some interesting observations in the percentage cell viability data of l-prolinamides 4a–4w, with each compound exhibiting inhibitory effects against one or more of the human cancer cell lines assayed. Most of the compounds examined showed that the percentage cell viability of the carcinoma cell lines decreased with increase in prolinamide concentration except 4f against SGC7901 and HepG2, and 4h against HepG2 and A549. Similar suspected hormetic responses [30] were detected for 4a, 4b, 4m and 4r against SGC7901 as well as 4j and 4t (against HepG2) and 4k and 4v (against A549). In addition, cell proliferation [29] appears to have occurred in some of the cell lines, such as A549 (with 4f, 4k and 4m), SGC7901 (with 4b and 4d) and HCT-116 (with 4j and 4t), where the percentage cell viability was greater than 100%, over the three concentrations.

The inhibitory effects of 100 µM concentrations of l-prolinamides 4a–4w on the four cancer cell lines under study are compared in figure 2, with the aid of bar chart diagrams. In precis, many of the prolinamides tested herein induced more than or equal to 50% cell inhibition in one or more of the cancer cell lines assayed, thereby highlighting the use of this class of compounds in cancer therapy. This is exemplified by 4a, 4e, 4k, 4o, 4q, 4r, 4s, 4u and 4w on HepG2; 4a, 4e, 4s, 4u and 4w on A549; 4q, 4s, 4u and 4w on SGC7901 and 4a, 4q, 4u and 4w on HCT-116. Accordingly, the human liver (HepG2) and colon (HCT-116) carcinoma cell lines proved to be the most sensitive and resistant cell lines, respectively, to the prolinamides under investigation. Additionally, l-prolinamides 4u and 4w showed greater than 50% inhibitions for the four cell lines assayed, whereas 4a, 4q and 4s were similarly active against three of the cell lines, alluding to the broad-spectrum nature of these compounds’ anti-cancer activities.

Figure 2.

(a–d) In vitro cytotoxic activity (MTT assay) of l-prolinamides (100 µM) against four human carcinoma cell lines. (a) SGC7901, human gastric carcinoma cell line; (b) HCT-116, human colon carcinoma cell line; (c) HepG2, human liver carcinoma cell line; and (d) A549, human lung carcinoma cell line. 5-FU, 5-fluorouracil (positive control). Concentration of l-prolinamides, 100 µM. Data are given as mean ± s.d. (n = 3).

It is noteworthy that excellent tumour inhibitory activities were observed with l-prolinamide 4a giving percentage inhibitions of 95.45 ± 0.67% and 93.33 ± 1.36% against A549 and HCT-116, respectively, whereas 4u recorded 91.98 ± 1.54% (SGC7901) and 83.36 ± 1.70% (A549). These proved more potent than the control, 5-fluorouracil, with percentage inhibitions of 64.29 ± 2.09% (A549), 81.20 ± 0.08% (HCT-116) and 68.72 ± 5.83% (SGC7901), respectively. l-Prolinamide 4a was less potent against SGC7901 and HepG2 relative to the reference compound while 4u was less potent than and comparable to 5-fluorouracil against HepG2 and HCT-116, respectively (cf. figure 2).

It is interesting to note that, across cell lines, the tertiary prolinamides, exemplified by 4a, 4s and 4w, showed better anti-tumour properties than the secondary prolinamides. However, the cyclic tertiary prolinamides (4i and 4n) were the exceptions. This may be owing to the restrictive nature of their carboxamide bonds. It was also observed that the longer the N′-alkyl chain substituents of the prolinamides (4a versus 4o), the stronger the antiproliferative activity; in concurrence with previous reports, which suggest that the greater lipophilicity afforded by the longer chains enable greater cell uptake [31]. Besides, N′-cyclohexylprolinamide 4e was more toxic to the HepG2 cell line than N′-phenylprolinamide 4s and N′,N′-dicyclohexylprolinamide 4w, whereas 4e inhibited the A549 cell line better than 4w (cf. table 1 and figure 2). A plausible explanation for the aforementioned is that N′-cycloalkyl-substituted compounds exhibit greater antineoplastic activities than their N′-aryl-substituted analogues [32] and that N′,N′-dicycloalkyl-substitution (relative to N′-monosubstitution) does not necessarily lead to increased antiproliferative activity.

Furthermore, it can be inferred from the results of the MTT assays of the four cell lines, except SGC7901, that branching (4j versus 4o; table 1) can confer reduced inhibitory potency against cancer cell lines. This is corroborated by N′-doubly substituted compounds (4j versus 4v) where increased branching resulted in decreased inhibitory activities against the four cell lines, except A549 (figure 2). Considering the N′-phenyl-containing prolinamides (e.g. 4s; cf. tables 1 and 2), electron-withdrawing substituents on the phenyl ring (4u) led to increased cytotoxicity, whereas electron-donating ring substituents (4k) decreased cytotoxic activity. Conversely, N′-toluenesulfonylprolinamide 4h showed higher cytotoxicity than N′-benzenesulfonylprolinamide 4f in all assays except HCT-116. In addition, N′-benzylprolinamide 4q proved more potent than N′-phenylprolinamide 4s against SGC7901 and HCT-116 but less potent against HepG2 and A549 (figure 2). Moreover, the substitution of 4-H on the prolyl ring by 4-OH (4k versus 4m) appeared to have had minimal effects on the cell lines, as revealed by the percentage cell viabilities of SGC7901, HCT-116 and HepG2, which were higher for 4m; except against A549 where the converse was the case. l-Prolinamides 4k and 4m are also both suspected of proliferating, rather than inhibiting, the human lung carcinoma cell line (A549).

Finally, the l-prolinamides 4a–4w synthesized and assayed in this protocol showed antiproliferative activities against one or more of the human carcinoma cell lines; with the most activity against HepG2 and the least activity against HCT-116. It is surprising, however, that none of the compounds bearing the aryl sulfonamide moiety (4f and 4h) showed a more than or equal to 50% inhibition against any of the four cancer cell lines assayed since sulfonamides have been reported to exhibit substantial in vitro and in vivo anti-tumour activities [33]. It is also instructive to note that none of the prolinamides 4a–4w exhibited a stronger cytotoxicity than 5-fluorouracil against the human liver carcinoma cell line (HepG2). The percentage cell viability of the nitrile, N′-(2″-cyanophenyl)-N-(4′-nitrophenyl)-l-prolinamide 4r against the four carcinoma cell lines tested ranged from 38.20 ± 6.03% (against HepG2) to 99.20 ± 3.57% (against HCT-116), as shown in table 2.

3. Conclusion

N′-substituted-N-(4′-nitrophenyl)-l-prolinamides 4a–4w have been successfully synthesized, characterized and assayed as potential anti-cancer agents. The synthetic route to the l-prolinamides was a facile and economical two-step protocol, starting from readily available reagents, to give the target carboxamides in 20–80% yield. The cytotoxicity of the resulting l-prolinamides were also investigated in vitro against the human gastric carcinoma (SGC7901), human colon carcinoma (HCT-116), human liver carcinoma (HepG2) and human lung carcinoma (A549) cell lines, using the MTT colorimetric assay.

Almost 50% of l-prolinamides 4a–4w showed strong anti-tumour activity against HepG2 but none was as potent as the positive control, 5-fluorouracil. However, 4a possessed stronger cancer inhibitory properties against the HCT-116 and A549 than 5-fluorouracil, whereas 4u was comparable in antineoplastic activities to the reference compound against HCT-116 but more potent against SGC7901 and A549. Of the 23 l-prolinamides assayed, N′,N′-dibutyl-N-(4′-nitrophenyl)-l-prolinamide 4a and N,N′-bis(4′-nitrophenyl)-l-prolinamide 4u showed the lowest percentage cell viability data across the four assayed cell lines and therefore possess the most promising cancer cells' inhibitory activities. Consequently, l-prolinamides 4a and 4u can be considered as potential broad-spectrum anti-cancer agents. Likewise, some of the other l-prolinamides are good lead compounds for structural optimization to develop potent antiproliferative agents for liver carcinomas.

4. Experimental procedure

Commercially available analytical grade reagents were used as purchased without further purification. Glassware was flame-dried and reactions carried out under an inert (dry nitrogen gas) atmosphere. Reactions were monitored by thin layer chromatography (TLC) on Merck silica gel 60 F₂₅₄ precoated plates using an ethyl acetate/n-hexane (1 : 2) solvent system and visualized under ultraviolet lamp (254 nm). Column chromatography was performed with silica gel (300–400 mesh) and solvents as indicated. Melting points were determined on a MEL-TEMP® capillary melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin Elmer Universal (ATR Spectrum 100) Fourier-transform-infrared spectrometer. ¹H-NMR (400 MHz & 600 MHz) and ¹³C-NMR (150 MHz) spectra were recorded on a Varian-Inova (400 MHz or 600 MHz) spectrometer with CDCl₃ or DMSO-d₆ as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) and coupling constants (J) are reported in parts per million (ppm), downfield from TMS and hertz (Hz), respectively. High-resolution mass spectra (m/z) were recorded on a micro TOF–QIII (ESI) spectrometer.

5. Chemistry

5.1. General procedure for the synthesis of N-(4′-substituted phenyl)-l-proline adducts

Into a clean round-bottomed flask was added l-proline 2 (12 mmol) in 1 : 1 ethanol/water (20 ml) and K₂CO₃ (3.5 equivalents), with stirring. The mixture was refluxed for 10 min before the addition of 4-substituted fluorobenzene 1 (10 mmol). The reaction mixture was then refluxed for 3h before cooling to ambient temperature, concentrating in vacuo and extracting into CH₂Cl₂ (10 ml). The aqueous layer was then acidified with 6M HCl and extracted into CH₂Cl₂ (20 ml × 3). The resulting organic layer was then washed with saturated brine solution (20 ml), dried over anhydrous Na₂SO₄, filtered and concentrated, under pressure, to give 3 as a solid.

5.2. Spectroscopic data

N-(4′-nitrophenyl)-l-proline (3a): yellow crystals (2.56 g, 90%); m.p. 160–161°C; IR (neat) ν_max (cm^–1): 3050, 2965, 2871, 1714, 1597, 1513, 1304, 1186, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 10.52 (1H, s, –COOH), 8.10 (2H, d, J = 9.1 Hz, ArH), 6.49 (2H, d, J = 9.1 Hz, ArH), 4.39 (1H, d, J = 7.0 Hz, –CHCOOH), 3.69–3.55 (1H, m, –CH_aH_bN–), 3.47 (1H, dd, J = 16.8, 8.4 Hz, –CH_aH_bN–), 2.47–2.28 (2H, m, –CH₂CH₂–), 2.25–2.09 (2H, m, –CH₂CH₂–);¹³C-NMR (150 MHz, CDCl₃) δ: 178.4 (–COOH), 151.0; 137.6; 126.2; 111.1 (ArH), 60.5 (–CHCOOH), 48.6 (–CH₂N–), 30.8 (–CH₂CH₂–), 23.6 (–CH₂CH₂–); HRMS (ESI) calculated for C₁₁H₁₂N₂O₄ [M + H]⁺ 237.0875, found 237.0879.

N-(4-cyanophenyl)-l-proline (3b): off-white crystals (1.79 g, 69%); m.p. 151–152°C; IR (neat) ν_max (cm^–1): 3463, 3040, 2949, 2857, 2208, 1727, 1600, 1215, 1172; ¹H-NMR (400 MHz, CDCl₃) δ: 10.31 (1H, s, –COOH), 7.45 (2H, d, J = 8.1 Hz, ArH), 6.52 (2H, d, J = 8.1 Hz, ArH), 4.30 (1H, d, J = 7.9 Hz, –CHCOOH), 3.56 (1H, d, J = 5.9 Hz, –CH_aH_bN–), 3.40 (1H, dd, J = 16.2, 8.0 Hz, –CH_aH_bN–), 2.40–2.30 (2H, m, –CH₂CH₂–), 2.22–2.12 (2H, m, –CH₂CH₂–);¹³C-NMR (150 MHz, CDCl₃) δ: 178.6 (–COOH), 149.3; 133.8 (ArH), 120.5 (–CN), 112.2; 98.7 (ArH), 60.4 (–CHCOOH), 48.4 (–CH₂N–), 30.9 (–CH₂CH₂–), 23.7 (–CH₂CH₂–); HRMS (ESI) calculated for C₁₂H₁₂N₂O₂ [M + H]⁺ 217.0977, found 217.0991.

4-Hydroxy-N-(4′-nitrophenyl)-l-proline (3c): yellow crystals (2.69 g, 89%); m.p. 152–153°C; IR (neat) ν_max (cm^–1): 3352, 3095, 2939, 1734, 1580, 1511, 1283, 1172, 1104, 1075; ¹H-NMR (400 MHz, DMSO-d₆) δ: 8.59 (2H, d, J = 9.0 Hz, ArH), 7.09 (2H, d, J = 9.0 Hz, ArH), 5.82 (1H, s, prolyl 4-OH), 4.99 (2H, s, –CHCOOH; –CHOH), 4.23–4.19 (1H, m, –CH_aH_bN–), 3.89–3.86 (1H, m, –CH_aH_bN–), 2.84–2.81 (1H, m, –CHCH_aH_b–), 2.77–2.50 (1H, m, –CHCH_aH_b–);¹³C-NMR (150 MHz, DMSO-d₆) δ: 173.7 (–COOH), 151.9; 136.5; 126.0; 111.4 (ArH), 68.2 (–CHCOOH), 59.5 (–CHOH), 56.8 (–CH₂N–), 38.9 (–CHCH₂–); HRMS (ESI) calculated for C₁₁H₁₂N₂O₅ [M + H]⁺ 253.0824, found 253.0825.

5.3. General procedure for the synthesis of N-(4′-substituted phenyl)-l-prolinamides

An oven-dried, N₂-evacuated round-bottomed flask equipped with a magnetic stirring bar was charged with N-(4′-substituted phenyl)-l-proline 3 (5 mmol) in CH₂Cl₂ (20 ml), SOCl₂ (2 ml, 27.6 mmol) and dimethyl formamide (one drop) and stirred for 3–4 h at ambient temperature. The reaction mixture was then concentrated to the crude acid chloride 5, which was taken up in CH₂Cl₂ (20 ml) and concentrated in vacuo to remove excess SOCl₂. The acid chloride was dissolved in CH₂Cl₂ (20 ml) and cooled to 0°C, under N₂, and NEt₃ (1.5 equivalents) was added. The amine (1.5 equivalents) was then added, with the aid of a dropping funnel and the reaction was left to stir overnight at ambient temperature. To work-up, the reaction mixture was acidified with 2M HCl (pH = 2) and the organic layer was washed with saturated brine solution (10 ml), dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum to a paste, which was purified via column chromatography on silica gel using n-hexane and ethyl acetate (2 : 1) as mobile phase.

5.4. Spectroscopic data

N′,N′-dibutyl-N-(4′-nitrophenyl)-l-prolinamide (4a): dark orange solid (1.04 g, 60%); m.p. 74–75°C; IR (neat) ν_max (cm^–1): 3090, 2954, 2928, 2870, 2852, 1638, 1596, 1579, 1518, 1484, 1296, 1108; ¹H-NMR (400 MHz, CDCl₃), Rotamers 1:1, δ: 8.08 (2H, d, J = 9.1 Hz, ArH), 6.36 (2H, d, J = 8.6 Hz, ArH), 4.56 (1H, d, J = 7.0 Hz, –NCHCON′–), 3.69 (1H, td, J = 9.2, 3.3 Hz, –CH_aH_bN–), 3.52 (1H, d, J = 8.7 Hz, –CH_aH_bN–), 3.46–3.36 (2H, m, –N′CH₂(CH₂)₂CH₃), 3.30 (2H, dd, J = 13.5, 6.9 Hz, –N′CH₂(CH₂)₂CH₃), 2.41–2.33 (m, 1H, –CH₂CH_aH_bCH–), 2.22 (1H, d, J = 7.6 Hz, –CH₂CH_aH_bCH–), 2.14–2.04 (2H, m, –CH₂CH_aH_bCH–), 1.71 (2H, dt, J = 15.5, 7.7 Hz, –N′CH₂CH₂CH₂CH₃), 1.55–1.37 (4H, m, –N′CH₂CH₂CH₂CH₃; –N′(CH₂)₂CH₂CH₃), 1.28 (2H, dd, J = 15.7, 8.2 Hz, –N′(CH₂)₂CH₂CH₃), 1.02 (3H, t, J = 7.3 Hz, –N′(CH₂)₃CH₃), 0.91 (3H, t, J = 7.3 Hz, –N′(CH₂)₃CH₃); ¹³C-NMR (150 MHz, CDCl₃), Rotamers 1:1, δ: 170.6 (–CON′–), 151.3; 137.6; 126.2; 110.8 (ArH), 59.1 (–CHCON′–), 49.0 (–CH₂N–), 47.3; 46.0 (–N′CH₂(CH₂)₂CH₃), 31.4; 31.1 (–N′CH₂CH₂CH₂CH₃), 29.6 (–CH₂CH₂CH–), 23.4 (–CH₂CH₂CH–), 20.21; 20.15 (–N′(CH₂)₂CH₂CH₃), 13.82; 13.78 (–N′(CH₂)₃CH₃); HRMS (ESI) calculated for C₁₉H₂₉N₃O₃ [M + H]⁺ 348.2287, found 348.2282.

N′-butyl-N-(4′-nitrophenyl)-l-prolinamide (4b): yellow solid (0.70 g, 48%); m.p. 145–147°C; IR (neat) ν_max (cm^–1): 3280, 3085, 2930, 2864, 1650, 1597, 1514, 1484, 1305, 1240, 1188, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 8.07 (2H, d, J = 9.3 Hz, ArH), 6.54 (2H, d, J = 9.3 Hz, ArH), 6.13 (1H, t, J = 5.3 Hz, –CONH), 4.18–4.14 (1H, m, –NCHCON′–), 3.71 (1H, ddd, J = 9.7, 7.7, 2.1 Hz, –N′CH_aH_b(CH₂)₂CH₃), 3.38 (1H, td, J = 9.9, 6.7 Hz, –N′CH_aH_b(CH₂)₂CH₃), 3.23 (2H, td, J = 7.2, 1.7 Hz, –CH₂N–), 2.35–2.27 (2H, m, –CH₂CH₂CH–), 2.16–1.98 (2H, m, –CH₂CH₂CH–), 1.45–1.36 (2H, m, –N′CH₂CH₂CH₂CH₃), 1.24 (2H, t, J = 7.3 Hz, –N′(CH₂)₂CH₂CH₃), 0.85 (3H, t, J = 7.3 Hz, –N′(CH₂)₃CH₃); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.7 (–CON′–), 151.7; 138.3; 126.0; 111.7 (ArH), 64.0 (–CHCON′–), 49.5 (–CH₂N–), 39.2 (–N′CH₂(CH₂)₂CH₃), 31.5 (–N′CH₂CH₂CH₂CH₃), 31.4 (–CH₂CH₂CH–), 23.9 (–CH₂CH₂CH–), 19.9 (–N′(CH₂)₂CH₂CH₃), 13.6 (–N′(CH₂)₃CH₃); HRMS (ESI) calculated for C₁₅H₂₁N₃O₃ [M + Na]⁺ 314.1481, found 314.1485.

N-(4′-nitrophenyl)-N′-propyl-l-prolinamide (4c): orange solid (0.68 g, 49%); m.p. 179–180°C; IR (neat) ν_max (cm^–1): 3277, 3084, 2963, 2928, 2871, 1649, 1597, 1554, 1513, 1483, 1305, 1238, 1186, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 8.06 (2H, d, J = 9.3 Hz, ArH), 6.54 (2H, d, J = 9.3 Hz, ArH), 6.18 (1H, s, –CONH), 4.20–4.12 (1H, m, –NCHCON′–), 3.77–3.66 (1H, m, –N′CH_aH_bCH₂CH₃), 3.38 (1H, td, J = 9.8, 6.8 Hz, –N′CH_aH_bCH₂CH₃), 3.27–3.13 (2H, m, –CH₂N–), 2.35–2.26 (2H, m, –CH₂CH₂CH–), 2.16–1.96 (2H, m, –CH₂CH₂CH–), 1.50–1.38 (2H, m, N′CH₂CH₂CH₃), 0.81 (3H, t, J = 7.4 Hz, N′CH₂CH₂CH₃);¹³C-NMR (150 MHz, CDCl₃) δ: 171.7 (–CON′–), 151.6; 138.4; 126.0; 111.8 (ArH), 64.0 (–CHCON′–), 49.5 (–CH₂N–), 41.1 (–N′CH₂CH₂CH₃), 31.5 (–CH₂CH₂CH–), 23.9 (–CH₂CH₂CH–), 22.8 (–N′CH₂CH₂CH₃), 11.2 (–N′CH₂CH₂CH₃); HRMS (ESI) calculated for C₁₄H₁₉N₃O₃ [M + H]⁺ 278.1505, found 278.1503.

N-(4′-nitrophenyl)-N′-(1″-prop-1″-ynyl)-l-prolinamide (4d): yellow solid (0.74 g, 54%); m.p. 158–159°C; IR (neat) ν_max (cm^–1): 3289, 3070, 2974, 2924, 2870, 1651, 1578, 1535, 1511, 1477, 1432, 1290, 1231, 1156, 1113; ¹H-NMR (400 MHz, CDCl₃) δ: 8.04 (2H, d, J = 9.3 Hz, ArH), 6.52 (2H, d, J = 9.3 Hz, ArH), 6.47 (1H, t, J = 5.3 Hz, –CONH), 4.21–4.16 (1H, m, –NCHCON′–), 4.06–4.01 (2H, m, –CH₂N–), 3.74 (1H, ddd, J = 9.6, 7.6, 2.2 Hz, –CH₂CH_aH_bCH–), 3.38 (1H, td, J = 9.8, 6.8 Hz, –CH₂CH_aH_bCH–), 2.36–2.28 (2H, m, propynyl –CH₂-H), 2.18 (1H, t, J = 2.5 Hz, propynyl –CH₂-H), 2.15–2.00 (2H, m, –CH₂CH_aH_bCH–); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.8 (–CON′–), 151.6; 138.6; 126.1; 111.8 (ArH), 79.0 (N′CCCH₃), 71.6 (–CHCON′–), 63.8 (N′CCCH₃), 49.5 (–CH₂N–), 31.4 (–CH₂CH₂CH–), 29.1 (–CH₂CH₂CH–), 23.9 (N′CCCH₃); HRMS (ESI) calculated for C₁₄H₁₅N₃O₃ [M + Na]⁺ 296.1011, found 296.0984.

N′-cyclohexyl-N-(4′-nitrophenyl)-l-prolinamide (4e): pale yellow solid (0.68 g, 43%); m.p. 252–253°C; IR (neat) ν_max (cm^–1): 3267, 3084, 2925, 2853, 1649, 1598, 1515, 1485, 1440, 1309, 1190, 1113;¹H-NMR (400 MHz, CDCl₃) δ: 8.06 (2H, d, J = 9.3 Hz, ArH), 6.53 (2H, d, J = 9.3 Hz, ArH), 5.94 (1H, d, J = 8.4 Hz, –CONH), 4.12 (1H, dd, J = 7.8, 3.7 Hz, –NCHCON′–), 3.73 (2H, dddd, J = 11.9, 9.7, 7.3, 3.3 Hz, –HN′CH–), 3.37 (1H, td, J = 9.8, 6.7 Hz, –CH₂N–), 2.36–2.24 (2H, m, prolyl –CH₂CH₂CH–), 2.15–1.97 (2H, m, cyclohexyl –CH₂–), 1.88–1.72 (2H, m, prolyl –CH₂CH₂CH–), 1.59 (3H, m, cyclohexyl –CH₂–), 1.37–1.26 (2H, m, cyclohexyl –CH₂–), 1.12–0.92 (3H, m, cyclohexyl –CH₂–);¹³C-NMR (150 MHz, CDCl₃), Rotamers (1:1), δ: 170.7 (–CON′–), 151.6; 138.4; 126.0; 111.8 (ArH), 64.0 (–CHCON′–), 49.5 (–CH₂N–), 48.2 (–HN′CH–), 33.0; 32.8 (cyclohexyl –CHCH₂–), 31.5 (prolyl –CH₂CH₂CH–), 29.6 (cyclohexyl –CH₂CH₂CH₂–), 25.3 (prolyl –CH₂CH₂CH–), 24.8; 24.7 (cyclohexyl –CH₂CH₂CH₂–); HRMS (ESI) calculated for C₁₇H₂₃N₃O₃ [M + H]⁺ 318.1818, found 318.1794.

N-(4′-nitrophenyl)-N′-(phenylsulfonyl)-l-prolinamide (4f): yellow solid (0.80 g, 58%); m.p. 189–191°C; IR (neat) ν_max (cm^–1): 3226, 3080, 2990, 2880, 1716, 1601, 1509, 1479, 1410, 1313, 1115; ¹H-NMR (400 MHz, CDCl₃) δ: 9.13 (1H, s, –CONH–), 7.85 (2H, d, J = 9.3 Hz, ArH), 7.81–7.76 (2H, m, ArH), 7.70 (1H, t, J = 7.5 Hz, ArH), 7.50 (2H, t, J = 7.9 Hz, ArH), 6.24 (2H, d, J = 9.2 Hz, ArH), 4.03 (1H, dd, J = 9.2, 3.2 Hz, –NCHCON′–), 3.81–3.75 (1H, m, –CH_aH_bN–), 3.31 (1H, dd, J = 16.5, 8.9 Hz, –CH_aH_bN–), 2.39–2.30 (1H, m, –CH₂CH_aH_bCH–), 2.26–2.18 (1H, m, –CH₂CH_aH_bCH–), 2.12–2.05 (2H, m, –CH₂CH_aH_bCH–); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.4 (–CON′–), 150.9; 138.9; 137.5; 134.5; 129.0; 128.0; 125.8; 111.8 (ArH), 63.9 (–CHCON′–), 49.6 (–CH₂N–), 31.3 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₇H₁₇N₃O₅S [M + Na]⁺ 398.0787, found 398.0775.

N′-(tert-butyl)-N-(4′-nitrophenyl)-l-prolinamide (4g): yellow solid (1.05 g, 72%); m.p. 141–142°C; IR (neat) ν_max (cm^–1): 3296, 3052, 2967, 1657, 1595, 1548, 1518, 1480, 1396, 1296, 1193, 1108; ¹H-NMR (400 MHz, CDCl₃) δ: 8.07 (2H, d, J = 9.3 Hz, ArH), 6.55 (2H, d, J = 9.3 Hz, ArH), 5.97 (1H, s, –CONH), 4.06 (1H, dd, J = 8.7, 2.8 Hz, –NCHCON′–), 3.75–3.69 (1H, m, –CH_aH_bN–), 3.40 (1H, dt, J = 16.5, 8.4 Hz, –CH_aH_bN–), 2.36–2.22 (2H, m, –CH₂CH₂CH–), 2.11 (2H, td, J = 6.8, 3.5 Hz, –CH₂CH₂CH–), 1.31 (9H, s, t-butyl –CH₃);¹³C-NMR (150 MHz, CDCl₃) δ: 170.9 (–CON′–), 151.6; 138.6; 126.0; 111.8 (ArH), 64.6 (–CHCON′–), 51.3 (–N′C(CH₃)₃), 49.5 (–CH₂N–), 31.4 (–CH₂CH₂CH–), 28.6 (–CH₂CH₂CH–), 23.9 (–N′C(CH₃)₃); HRMS (ESI) calculated for C₁₅H₂₁N₃O₃ [M + Na]⁺ 314.1481, found. 314.1463.

N-(4′-nitrophenyl)-N′-(4″-tosyl)-l-prolinamide (4h): yellow solid (0.99 g, 51%); m.p. 93–95°C; IR (neat) ν_max (cm^–1): 3237, 3025, 2905, 2872, 1719, 1596, 1492, 1296, 1172, 1110, 1083; ¹H-NMR (600 MHz, CDCl₃) δ: 9.34 (1H, s, –CONH), 7.76 (2H, d, J = 9.1 Hz, ArH), 7.57 (2H, d, J = 8.2 Hz, ArH), 7.24 (2H, d, J = 8.1 Hz, ArH), 6.18 (2H, d, J = 9.0 Hz, ArH), 4.01 (1H, dd, J = 9.2, 3.0 Hz, –NCHCON′–), 3.79–3.74 (1H, m, –CH_aH_bN–), 3.28 (1H, q, J = 8.3 Hz, –CH_aH_bN–), 2.47 (3H, s, tosyl –CH₃), 2.37–2.31 (1H, m, –CH₂CH_aH_bCH–), 2.20 (1H, ddd, J = 13.1, 8.3, 4.9 Hz, –CH₂CH_aH_bCH–), 2.10–2.04 (2H, m, –CH₂CH_aH_bCH–);¹³C-NMR (150 MHz, CDCl₃) δ: 171.6 (–CON′–), 150.9; 146.0; 138.7; 134.5; 129.5; 128.0; 125.7; 111.7 (ArH), 63.8 (–CHCON′–), 49.5 (–CH₂N–), 31.4 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–), 21.7 (ArCH₃); HRMS (ESI) calculated for C₁₈H₁₉N₃O₅S [M + Na]⁺ 412.0948, found 412.0938.

4″-Morpholinyl N-(4′-nitrophenyl)-2-pyrrolidinyl ketone (4i): yellow solid (0.69 g, 45%); m.p. 214–216°C; IR (neat) ν_max (cm^–1): 2913, 2886, 2863, 2813, 1639, 1595, 1517, 1476, 1433, 1287, 1234, 1199, 1107, 1034; ¹H-NMR (400 MHz, CDCl₃) δ: 8.07 (2H, d, J = 9.4 Hz, ArH), 6.35 (2H, d, J = 9.2 Hz, ArH), 4.61 (1H, dd, J = 8.6, 2.4 Hz, –NCHCON′–), 3.85–3.48 (10H, m, morpholinyl –CH₂–; –CH₂N–), 2.42–2.31 (1H, m, –CH₂CH_aH_bCH–), 2.23–2.02 (3H, m, –CH₂CH_aH_bCH–; –CH₂CH_aH_bCH–);¹³C-NMR (150 MHz, CDCl₃) δ: 169.6 (–CON′–), 151.1; 137.4; 126.2; 110.9 (ArH), 67.0; 66.5 (–CH₂O–), 58.9 (–CHCON′–), 48.8 (–CH₂N–); 45.8; 42.5 (–N′CH₂–), 30.5 (–CH₂CH₂CH–), 23.4 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₅H₁₉N₃O₄ [M + Na]⁺ 328.1273, found 328.1274.

N′,N′-diisopropyl-N-(4′-nitrophenyl)-l-prolinamide (4j): yellow solid (0.45 g, 28%); m.p. 159–160°C; IR (neat) ν_max (cm^–1): 3087, 3015, 1596, 1507, 1309, 1111; ¹H-NMR (400 MHz, CDCl₃), Rotamers (1:1), δ: 8.06 (2H, d, J = 8.8 Hz, ArH), 6.34 (2H, d, J = 7.0 Hz, ArH), 4.52 (1H, d, J = 8.0 Hz, –NCHCON′–), 4.11 (1H, dt, J = 12.7, 6.2 Hz, –N′CH(CH₃)₂), 3.65 (1H, d, J = 5.4 Hz, N′CH(CH₃)₂), 3.52 (1H, dd, J = 16.2, 8.0 Hz, –CH_aH_bN–), 3.43 (1H, dt, J = 12.7, 6.2 Hz, –CH_aH_bN–), 2.41–2.29 (1H, m, –CH₂CH_aH_bCH–), 2.21 (1H, dt, J = 16.4, 8.1 Hz, –CH₂CH_aH_bCH–), 2.12–1.98 (2H, m, –CH₂CH_aH_bCH–), 1.43–1.23 (12H, m, –N′CH(CH₃)₂); ¹³C-NMR (150 MHz, CDCl₃), Rotamers (4:3), δ: 169.3 (–CON′–), 151.4; 137.0; 126.1; 110.7 (ArH), 60.2 (–CHCON′–), 48.9; 48.4 (–N′CH(CH₃)₂), 46.4 (–CH₂N–), 30.7 (–CH₂CH₂CH–), 23.4 (–CH₂CH₂CH–), 21.2; 20.6; 20.53; 20.45 (–N′CH(CH₃)₂); HRMS (ESI) calculated for C₁₇H₂₅N₃O₃ [M + H]⁺ 320.1974, found 320.1969.

N-(4′-nitrophenyl)-N′-(4″-tolyl)-l-prolinamide (4k): yellow solid (1.39 g, 80%); m.p. 218–220°C; IR (neat) ν_max (cm^–1): 3291, 2913, 2958, 1667, 1598, 1537, 1513, 1480, 1297, 1182, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 8.06 (1H, s, –CONH), 8.01 (2H, d, J = 9.2 Hz, ArH), 7.33 (2H, d, J = 8.4 Hz, ArH), 7.07 (2H, d, J = 8.2 Hz, ArH), 6.57 (2H, d, J = 9.3 Hz, ArH), 4.24 (1H, t, J = 5.8 Hz, –NCHCON′–), 3.83–3.76 (1H, m, –CH_aH_bN–), 3.40 (1H, dd, J = 17.3, 8.8 Hz, –CH_aH_bN–), 2.41–2.33 (2H, m, –CH₂CH₂CH–), 2.28 (3H, s, ArCH₃), 2.14 (2H, dd, J = 13.8, 7.0 Hz, –CH₂CH₂CH–);¹³C-NMR (150 MHz, CDCl₃) δ: 170.3 (–CON′–), 151.7; 138.8; 134.8; 134.2; 129.5; 126.1; 120.4; 112.0 (ArH), 64.5 (–CHCON′–), 49.8 (–CH₂N–), 31.6 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–), 20.9 (ArCH₃); HRMS (ESI) calculated for C₁₈H₁₉N₃O₃ [M + Na]⁺ 348.1324, found 348.1341.

N-(4′-cyanophenyl)-N′-(4″-tolyl)-l-prolinamide (4l): brown oil (0.34 g, 21%); IR (neat) ν_max (cm^–1): 3303, 2920, 2868, 2211, 1666, 1602, 1511, 1364, 1309, 1174; ¹H-NMR (600 MHz, CDCl₃) δ: 7.93 (1H, s, –CONH), 7.48 (2H, d, J = 8.8 Hz, ArH), 7.32 (2H, d, J = 8.4 Hz, ArH), 7.09 (2H, d, J = 8.2 Hz, ArH), 6.67 (2H, d, J = 8.8 Hz, ArH), 4.17 (1H, dd, J = 8.6, 3.1 Hz, –NCHCON′–), 3.80–3.76 (1H, m, –CH_aH_bN–), 3.36 (1H, td, J = 9.7, 6.8 Hz, –CH_aH_bN–), 2.41–2.33 (2H, m, –CH₂CH₂CH–), 2.29 (3H, s, ArCH₃), 2.16–2.05 (2H, m, –CH₂CH₂CH–);¹³C-NMR (150 MHz, CDCl₃) δ: 170.6 (–CON′–), 150.0; 134.6; 134.3; 133.7; 129.5 (ArH), 120.3 (–CN), 113.2; 100.4 (ArH), 64.6 (–CHCON′–), 49.6 (–CH₂N–), 31.6 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–), 20.9 (ArCH₃); HRMS (ESI) calculated for C₁₉H₁₉N₃O [M + Na]⁺ 328.1426, found 328.1423.

trans-4-hydroxy-N-(4′-nitrophenyl)-N′-(4″-tolyl)-l-prolinamide (4m): yellow solid (0.36 g, 21%); m.p. 236–238°C; IR (neat) ν_max (cm^–1): 3297, 2952, 2918, 1659, 1596, 1493, 1311, 1196, 1113; ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.26 (1H, s, –CONH), 7.11 (2H, d, J = 9.4 Hz, ArH), 6.50 (2H, d, J = 8.4 Hz, ArH), 6.15 (2H, d, J = 8.3 Hz, ArH), 5.65 (2H, d, J = 8.6 Hz, ArH), 4.40 (1H, s, –CHOH), 3.62 (1H, t, J = 7.4 Hz, –NCHCON′–), 3.56 (1H, dd, J = 7.5, 3.9 Hz, –CHOH), 2.84 (1H, dd, J = 10.7, 4.8 Hz, –CH_aH_bN–), 1.56–1.55 (1H, m, –CH_aH_bN–), 1.44–1.39 (1H, m, –CHCH_aH_bCH–), 1.29 (3H, s, ArCH₃), 1.25–1.20 (1H, m, –CHCH_aH_bCH–); ¹³C-NMR (150 MHz, DMSO-d₆) δ: 170.5 (–CON′–), 152.3; 136.6; 136.4; 133.2; 129.5; 126.3; 120.1; 111.7 (ArH), 68.6 (–CHCON′–), 61.6 (–CHOH), 57.8 (–CH₂N–), 40.0 (–CHCH₂CH–), 20.9 (ArCH₃); HRMS (ESI) calculated for C₁₈H₁₉N₃O₄ [M + H]⁺ 342.1454, found 342.1444.

2″-Isoindolinyl-N-(4′-nitrophenyl)-2-pyrrolidinyl ketone (4n): yellow solid (0.76 g, 45%); m.p. 215–217°C; IR (neat) ν_max (cm^–1): 3075, 3060, 2953, 2854, 1654, 1581, 1512, 1479, 1288, 1195, 1106; ¹H-NMR (400 MHz, CDCl₃) δ: 8.15 (1H, d, J = 8.0 Hz, indolinyl ArH), 8.02 (2H, d, J = 9.0 Hz, ArH), 7.23 (1H, d, J = 7.2 Hz, indolinyl ArH), 7.15 (1H, t, J = 7.6 Hz, indolinyl ArH), 7.05 (1H, t, J = 7.3 Hz, indolinyl ArH), 6.37 (2H, d, J = 8.3 Hz, ArH), 4.56 (1H, d, J = 7.9 Hz, –NCHCON′–), 4.35 (1H, dd, J = 17.3, 9.7 Hz, indolinyl –N′CH_aH_b–), 4.17 (1H, dd, J = 16.5, 9.8 Hz, indolinyl –N′CH_a′H_b–), 3.74 (1H, t, J = 7.2 Hz, indolinyl –N′CH_aH_b–), 3.54 (1H, dd, J = 16.2, 7.8 Hz, indolinyl –N′CH_aH_b′–), 3.33 (2H, dd, J = 16.4, 8.4 Hz, prolyl –CH₂N–), 2.45 (1H, dd, J = 17.9, 8.8 Hz, –CH₂CH_aH_bCH–), 2.32–2.11 (3H, m, –CH₂CH_aH_bCH–);¹³C-NMR (150 MHz, CDCl₃) δ: 169.2 (–CON′–), 151.0; 142.7; 137.4; 130.8; 127.7; 126.3; 124.7; 124.4; 117.3; 110.9 (ArH), 61.0 (–CHCON′–), 49.0; 47.6 (indolinyl –N′CH₂–), 30.4 (prolyl –CH₂N–), 28.4 (–CH₂CH₂CH–), 23.1 (–CH₂CH₂CH-); HRMS (ESI) calculated for C₁₉H₁₉N₃O₃ [M + Na]⁺ 360.1324, found 360.1323.

N-(4′-nitrophenyl)-N′,N′-dipropyl-l-prolinamide (4o): yellow solid (0.77 g, 48%); m.p. 136–138°C; IR (neat) ν_max (cm^–1): 3025, 2961, 2922, 2873, 1637, 1597, 1519, 1488, 1300, 1230, 1109; ¹H-NMR (600 MHz, CDCl₃) δ: 8.06 (2H, d, J = 9.4 Hz, ArH), 6.35 (2H, d, J = 8.5 Hz, ArH), 4.57 (1H, dd, J = 8.5, 2.5 Hz, –NCHCON′–), 3.68 (1H, td, J = 9.0, 3.7 Hz, prolyl –CH_aH_bN–), 3.51 (1H, dd, J = 16.9, 7.7 Hz, prolyl –CH_aH_bN–), 3.43–3.33 (2H, m, –N′CH₂CH₂CH₃), 3.31–3.21 (2H, m, –N′CH₂CH₂CH₃), 2.38 (1H, ddd, J = 17.8, 8.2, 5.8 Hz, –CH₂CH_aH_bCH–), 2.25–2.17 (1H, m, –CH₂CH_aH_bCH–), 2.11–2.03 (2H, m, –CH₂CH_aH_bCH–), 1.75 (2H, dd, J = 14.9, 7.4 Hz, –N′CH₂CH₂CH₃), 1.55 (2H, dd, J = 15.0, 7.5 Hz, –N′CH₂CH₂CH₃), 1.02 (3H, t, J = 7.4 Hz, –N′CH₂CH₂CH₃), 0.86 (3H, t, J = 7.4 Hz, –N′CH₂CH₂CH₃);¹³C-NMR (150 MHz, CDCl₃) δ: 170.7 (–CON′–), 151.3; 137.1; 126.2; 110.8 (ArH), 59.1 (–CHCON′–), 49.2; 49.0 (–N′CH₂CH₂CH₃), 47.9 (–CH₂N–), 31.1 (–CH₂CH₂CH–), 23.4 (–CH₂CH₂CH–), 22.5; 20.8 (–N′CH₂CH₂CH₃), 11.4; 11.3 (–N′CH₂CH₂CH₃); HRMS (ESI) calculated for C₁₇H₂₅N₃O₃ [M + H]⁺ 320.1974, found 320.1998.

N-(4′-nitrophenyl)-N′-(2″-pyridinyl)-l-prolinamide (4p): yellow solid (0.72 g, 46%); m.p. 180–182°C; IR (neat) ν_max (cm^–1): 3290, 3080, 2980, 2876, 1708, 1595, 1515, 1475, 1333, 1290, 1197, 1110; ¹H-NMR (600 MHz, CDCl₃) δ: 8.50 (1H, d, J = 12.6 Hz, –CONH), 8.25 (1H, d, J = 8.2 Hz, pyridinyl –CH–), 8.20 (1H, s, pyridinyl –CH–), 8.15–8.08 (2H, m, ArH), 7.74–7.69 (1H, m, pyridinyl –CH–), 7.05 (1H, dd, J = 7.3, 3.5 Hz, pyridinyl –CH–), 6.65–6.61 (2H, m, ArH), 4.30 (1H, dd, J = 8.6, 2.8 Hz, –NCHCON′–), 3.85 (1H, dd, J = 7.4, 5.2 Hz, –CH_aH_bN–), 3.44 (1H, q, J = 8.8 Hz, –CH_aH_bN–), 2.48–2.36 (2H, m, –CH₂CH₂CH–), 2.15 (2H, dt, J = 13.6, 6.9 Hz, –CH₂CH₂CH–);¹³C-NMR (150 MHz, CDCl₃) δ: 171.1 (–CON′–), 151.6 (ArH), 150.4; 147.8; 139.1 (pyr-ArH), 138.5; 126.1 (ArH), 120.4; 114.0 (pyr-ArH), 112.1 (ArH), 64.6 (–CHCON′–), 49.8 (–CH₂N–), 31.7 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₆H₁₆N₄O₃ [M + H]⁺ 313.1301, found 313.1312.

N′-benzyl-N-(4′-nitrophenyl)-l-prolinamide (4q): yellow solid (0.89 g, 55%); m.p. 178–179°C; IR (neat) ν_max (cm^–1): 3292, 3085, 2979, 2952, 2877, 1707, 1648, 1513, 1479, 1396, 1293, 1198, 1115; ¹H-NMR (400 MHz, CDCl₃) δ: 8.01 (2H, d, J = 8.9 Hz, ArH), 7.25 (1H, s, –CONH), 7.24–7.09 (4H, m, ArH), 6.51 (3H, m, ArH), 4.50–4.35 (2H, m, ArCH₂–), 4.21 (1H, t, J = 5.4 Hz, –NCHCON′–), 3.68 (1H, t, J = 7.9 Hz, –CH_aH_bN–), 3.35 (1H, dd, J = 16.4, 9.5 Hz, –CH_aH_bN–), 2.43–2.29 (2H, m, –CH₂CH₂CH–), 2.05 (2H, dt, J = 20.3, 11.7 Hz, –CH₂CH₂CH–);¹³C-NMR (150 MHz, CDCl₃) δ: 171.9 (–CON′–), 151.8; 138.6; 137.8; 128.7; 127.6; 127.4; 126.0; 111.9 (ArH), 64.0 (–CHCON′–), 49.5 (–CH₂N–), 43.4 (ArCH₂–), 31.5 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₈H₁₉N₃O₃ [M + H]⁺ 326.1505, found 326.1510.

N′-(2″-cyanophenyl)-N-(4′-nitrophenyl)-l-prolinamide (4r): yellow solid (0.59 g, 35%); m.p. 211–213°C; IR (neat) ν_max (cm^–1): 3257, 3060, 2950, 2930, 2867, 2226, 1682, 1596, 1484, 1450, 1390, 1312, 1166, 1112; ¹H-NMR (400 MHz, CDCl₃) δ: 8.56 (1H, s, –CONH), 8.35 (1H, d, J = 8.4 Hz, ArH), 8.14 (2H, d, J = 9.1 Hz, ArH), 7.60 (1H, t, J = 7.8 Hz, ArH), 7.52 (1H, d, J = 7.6 Hz, ArH), 7.19 (1H, t, J = 7.6 Hz, ArH), 6.69 (2H, d, J = 9.1 Hz, ArH), 4.36 (1H, dd, J = 8.0, 3.0 Hz, –NCHCON′–), 4.00–3.89 (1H, m, –CH_aH_bN–), 3.49 (1H, dd, J = 17.4, 8.9 Hz, –CH_aH_bN–), 2.53–2.38 (2H, m, –CH₂CH₂CH–), 2.23 (2H, dt, J = 11.2, 5.8 Hz, –CH₂CH₂CH–); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.0 (–CON′–), 151.3; 139.5; 139.4; 134.2; 132.0; 126.2; 124.8; 121.0 (ArH), 115.8 (–CN), 112.3; 102.8 (ArH), 64.6 (–CHCON′–), 49.7 (–CH₂N–), 31.6 (–CH₂CH₂CH–), 24.0 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₈H₁₆N₄O₃ [M + H]⁺ 337.1301, found 337.1313.

N′-methyl-N-(4′-nitrophenyl)-N′-phenyl-l-prolinamide (4s): yellow solid (0.57 g, 33%); m.p. 186–188°C; IR (neat) ν_max (cm^–1): 3055, 3042, 2952, 2920, 2868, 1656, 1593, 1483, 1378, 1303, 1193, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 8.09 (2H, d, J = 8.8 Hz, ArH), 7.53 (2H, t, J = 7.3 Hz, ArH), 7.44 (1H, t, J = 7.2 Hz, ArH), 7.34 (2H, d, J = 7.5 Hz, ArH), 6.35 (2H, d, J = 8.7 Hz, ArH), 4.24 (1H, d, J = 5.9 Hz, –NCHCON′–), 3.72–3.63 (1H, m, –CH_aH_bN–), 3.44 (1H, dd, J = 15.8, 8.1 Hz, –CH_aH_bN–), 3.30 (3H, s, –N′CH₃), 2.34–1.94 (4H, m, –CH₂CH₂CH–); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.8 (–CON′–), 151.0; 142.8; 137.3; 130.3; 128.6; 127.2; 126.2; 110.8 (ArH), 59.1 (–CHCON′–), 49.1 (–CH₂N–), 37.9 (–N′CH₃), 31.5 (–CH₂CH₂CH–), 23.6 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₈H₁₉N₃O₃ [M + Na]⁺ 348.1324, found 348.1303.

N′-(2″-(hydroxymethyl)phenyl)-N-(4′-nitrophenyl)-l-prolinamide (4t): yellow solid (0.90 g, 53%); m.p. 179–180°C; IR (neat) ν_max (cm^–1): 3248, 3080, 2980, 2952, 2869, 1661, 1584, 1537, 1512, 1482, 1312, 1181, 1112; ¹H-NMR (600 MHz, CDCl₃) δ: 9.52 (1H, s, –CONH), 8.15 (1H, d, J = 8.2 Hz, ArH), 8.10 (2H, d, J = 9.1 Hz, ArH), 7.34–7.29 (1H, m, ArH), 7.09–7.02 (2H, m, ArH), 6.62 (2H, d, J = 9.2 Hz, ArH), 4.47 (1H, d, J = 12.6 Hz, –NCHCON′–), 4.30 (2H, dd, J = 11.9, 6.1 Hz, –CH₂OH), 3.87–3.82 (1H, m, –CH_aH_bN–), 3.47 (1H, dd, J = 16.8, 9.6 Hz, –CH_aH_bN–), 2.40 (2H, dd, J = 9.6, 5.7 Hz, –CH₂CH₂CH–), 2.15 (2H, dt, J = 12.3, 5.7 Hz, –CH₂CH₂CH–), 1.68 (1H, s, –CH₂OH);¹³C-NMR (150 MHz, CDCl₃) δ: 170.5 (–CON′–), 151.3; 138.5; 136.9; 129.2; 128.9; 128.6; 126.1; 124.5; 121.6; 111.8 (ArH), 64.6 (–CHCON′–), 64.4 (–CH₂OH), 49.2 (–CH₂N–), 31.5 (–CH₂CH₂CH–), 23.9 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₈H₁₉N₃O₄ [M + H]⁺ 342.1454, found 342.1471.

N,N′-bis(4′-nitrophenyl)-l-prolinamide (4u): yellow solid (0.78 g, 44%); m.p. 137–139°C; IR (neat) ν_max (cm^–1): 3318, 3085, 2921, 2869, 1687, 1595, 1499, 1292, 1158, 1108; ¹H-NMR (600 MHz, DMSO-d₆) δ: 9.95 (1H, s, –CONH), 7.33 (2H, d, J = 9.2 Hz, ArH), 7.18 (2H, d, J = 9.4 Hz, ArH), 6.97 (2H, d, J = 9.2 Hz, ArH), 5.72 (2H, d, J = 8.1 Hz, ArH), 3.68 (1H, dd, J = 8.8, 2.1 Hz, –NCHCON′–), 2.81–2.76 (1H, m, –CH_aH_bN–), 2.59 (1H, dd, J = 17.6, 8.1 Hz, –CH_aH_bN–), 1.57–1.49 (1H, m, –CH₂CH_aH_bCH–), 1.30 (1H, dt, J = 6.9, 5.7 Hz, –CH₂CH_aH_bCH–), 1.21–1.15 (2H, m, –CH₂CH₂CH–); ¹³C-NMR (150 MHz, DMSO-d₆) δ: 171.8 (–CON′–), 151.8; 145.2; 142.9; 136.8; 126.4; 125.3; 119.7; 111.8 (ArH), 62.6 (–CHCON′–), 49.3 (–CH₂N–), 31.7 (–CH₂CH₂CH–), 23.7 (–CH₂CH₂CH–); HRMS (ESI) calculated for C₁₇H₁₆N₄O₅ [M + Na]⁺ 379.1018, found 379.1007.

N′-isopropyl-N-(4′-nitrophenyl)-l-prolinamide (4v): yellow solid (0.69 g, 50%); m.p. 231–232°C; IR (neat) ν_max (cm^–1): 3275, 3081, 2972, 2870, 1647, 1597, 1548, 151, 1483, 1303, 1238, 1188, 1111; ¹H-NMR (400 MHz, CDCl₃) δ: 8.12 (2H, d, J = 9.0 Hz, ArH), 6.56 (2H, d, J = 9.0 Hz, ArH), 5.84 (1H, d, J = 7.6 Hz, –CONH), 4.16–4.04 (2H, m, –NCHCON′N′–; –N′CH(CH₃)₂), 3.72 (1H, t, J = 8.1 Hz, –CH_aH_bN–), 3.38 (1H, dd, J = 16.5, 9.6 Hz, –CH_aH_bN–), 2.31 (2H, dd, J = 13.1, 9.3 Hz, –CH₂CH₂CH–), 2.17–1.96 (2H, m, –CH₂CH₂CH–), 1.07 (6H, dd, J = 28.0, 6.5 Hz, –N′CH(CH₃)₂); ¹³C-NMR (150 MHz, CDCl₃) δ: 170.8 (–CON′–), 151.6; 138.7; 126.1; 111.8 (ArH), 64.1 (–CHCON′–), 49.6 (–CH₂N–), 41.5 (–N′CH(CH₃)₂), 31.4 (–CH₂CH₂CH–), 23.9 (–CH₂CH₂CH–), 22.7; 22.5 (–N′CH(CH₃)₂); HRMS (ESI) calculated for C₁₄H₁₉N₃O₃ [M + H]⁺ 278.1505, found 278.1494.

N′,N′-dicyclohexyl-N-(4′-nitrophenyl)-l-prolinamide (4w): yellow solid (0.60 g, 30%); m.p. 140–142°C; IR (neat) ν_max (cm^–1): 3031, 2926, 2851, 1648, 1596, 1512, 1484, 1289, 1234, 1182, 1108; ¹H-NMR (600 MHz, CDCl₃) δ: 8.07 (2H, d, J = 9.3 Hz, ArH), 6.36 (2H, d, J = 4.9 Hz, ArH), 4.52 (1H, dd, J = 8.6, 2.7 Hz, –NCHCON′–), 3.66 (1H, td, J = 8.9, 4.0 Hz, cyclohexyl –CH–), 3.61–3.51 (2H, m, cyclohexyl –CH–; –CH_aH_bN–), 2.39–2.32 (2H, m, –CH_aH_bN–; –CH₂CH_aH_bCH–), 2.24–2.16 (1H, m, –CH₂CH_aH_bCH–), 2.11–1.98 (2H, m, cyclohexyl –CH₂–), 1.91 (3H, m, –CH₂CH_aH_bCH–; cyclohexyl –CH_aH_b–), 1.74 (4H, m, cyclohexyl –CH_aH_b–; –CH₂–), 1.51–1.07 (13H, m, cyclohexyl –CH₂–); ¹³C-NMR (150 MHz, CDCl₃) δ: 169.5 (–CON′–), 151.3; 137.1; 126.1; 110.8 (ArH), 60.5 (–CHCON′–), 57.6; 56.7 (cyclohexyl –CH–), 49.0 (–CH₂N–), 31.6; 31.1 (cyclohexyl –CH₂–), 30.8 (prolyl –CH₂CH₂CH–), 30.0; 29.6 (cyclohexyl –CH₂–), 26.50 (prolyl –CH₂CH₂CH–), 26.48; 26.02; 25.97; 25.2; 25.1; 23.4 (cyclohexyl –CH₂–); HRMS (ESI) calculated for C₂₃H₃₃N₃O₃ [M + H]⁺ 400.2600, found 400.2604.

6. Biology

6.1. Materials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-fluorourasil and dimethyl sulfoxide (DMSO) were purchased from Sigma and Merck, respectively, whereas fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were both obtained from Gibco. Dulbecco's Modified Eagle Medium (DMEM) was purchased from Hyclone.

6.2. Cell lines and culture

Four human cancer cell lines: gastric carcinoma (SGC7901), colon carcinoma (HCT-116), liver carcinoma (HepG2) and lung carcinoma (A549) cell lines were used in this study. All the cell lines were grown as adherent monolayers in flasks with DMEM cultured media supplemented with 10% FBS and 1% streptomycin/penicillin in a humidified incubator with 5% CO₂ at 37°C.

6.3. MTT assay

The cytotoxicies of l-prolinamides 4a–4w were estimated against human gastric (SGC7901), colon (HCT-116), liver (HepG2) and lung (A549) cancer cell lines using the MTT assay. The cells were seeded evenly in 96-well plates with a cell density of 3000 cells well⁻¹ with FBS and incubated overnight for attachment. After 24 h, 100 µl (per well) of different concentrations of l-prolinamides 4a–4w in serial 10-fold dilutions (1 µM, 10 µM and 100 µM) were added in triplicates into the wells and incubated at 5% CO₂ at 37°C. Stock solutions of l-prolinamides 4a–4w were prepared in DMSO; followed by dilution in buffer so that the final concentration of DMSO in the culture media was 1%. After 72 h of incubation, the culture medium was carefully removed and MTT solution (100 µl) was added into each well and incubated at 37°C for 2 h, to allow the viable cells to bio-transform the yellow-coloured MTT into dark-blue formazan crystals. The MTT solution was then aspirated and DMSO (150 µl) was added into each well and incubated at 37°C at 800 r.p.m. for 10 min, to dissolve the formazan crystals. The optical density (absorbance) of each sample was measured at 490 nm using the M3 SpectraMax microplate reader (Molecular Devices).

The percentage cell viability and inhibition were calculated using equations (6.1) and (6.2), and were used as a measure of the compounds' inhibition potencies on the cancer cell lines:

$$\text{cell}\hspace{0.17em}\text{viability }(\text{\%})=\frac{\text{O}{\text{D}}_{\mathrm{sample}}}{\text{O}{\text{D}}_{\mathrm{control}}}\times 100\text{\%}$$ 6.1

and

$$\hspace{0.17em}\text{\%}\hspace{0.17em}\text{inhibition}=100\text{\%}\hspace{0.17em}\text{–}\hspace{0.17em}\text{cell}\hspace{0.17em}\text{viability }(\text{\%}).$$ 6.2

Data accessibility

NMR spectroscopic data for the synthesized compounds are included as the electronic supplementary material. Data are also deposited in the Dryad Digital Repository [34].

Authors' contributions

A.O. participated in the design of the study and carried out the synthesis; J.I. participated in the design of the study and data analyses, drafted and submitted the manuscript; X.B. coordinated the benchwork and reviewed the draft manuscript; O.A. participated in the design of the study and reviewed the draft manuscript; J.K. carried out the assays; C.G. coordinated the assays and reviewed the draft manuscript; O.F. led the conception and design of the study, coordinated the study and reviewed drafts. All authors approved the manuscript for publication and agree to be held accountable for the work performed therein.

Competing interests

There are no conflicts of interest to declare.

Funding

This work was funded, in part, by a University of Lagos Central Research Committee grant (CRC no. 2015/25).