1. Introduction
Among heterocyclic compounds, the 2-amino-4H-chromenes and their 4H-chromeno[2,3-d]pyrimidin derivatives represent very attractive products known for their broad spectrum of biological activities. They are referenced as potential anti-tumor agents [1] [2], anti-bacterial [3]-[5], anti-inflammatory agents [6] and also exhibited significant selective activity against bacteria and fungi [7]. It is interesting to point out that this type of structure is also present in natural products. As examples, 4H-chromene appears in Wisteria sinensis plant [8] and in the leaves of Calyptranthes tricona [9]. Among the 2-amino-4H-chromene products, those which have been a most advanced stage of pharmacological development are respectively drug-candidate LY290181 as potent antiproliferative compound blocking cells in G2/M phase of the cell cycle [10], CrolibulinTM (EPC 2407) in clinical phase I/II trials for treatment of aggressive solid tumors [11] and HA14.1 as tubulin inhibitor in pre-clinal and clinal phase I trials for cancer [12].
As an extension of our previous studies concerning the use of 2-amino-4H-chromene as starting platform [13] for the construction of fused heterocyclic systems with biological applications, we reported herein the synthesis of 4H-chromeno[2,3-d]pyrimidin-3(5H)-amine, explored in vitro the cytotoxic activities on a panel of tumoral cell lines and determined in silico their physicochemical and ADME (Absorption, Distribution, Metabolism, Excretion) properties.
2. Results and Discussion
2.1. Chemistry
The β-enaminonitrile moiety which is directly integrated in the 2-amino-4H-chromen-4-carbonitrile platform offers a wide diversity of molecular reactivity. The simultaneous presence of the carbonitrile function in position C-4 as electrophilic center and amino function in position C-2 as nucleophilic center allows heterocyclization reactions. The methodology adopted for the preparation of 2-amino-4H-chromen-4-carbonitrile 4(a-e) is outlined in Scheme 1 according to procedures reported in literature [13].
To enrich the molecular diversity of this platform, we initially used 2-hydroxy-3-methoxybenzaldehyde 1a or orthovanilin, 2-hydroxy-4-methoxybenzaldehyde 1b, 2-hydroxynaphtaldehyde 1c, 2-hydroxy-5-methoxybenzaldehyde 1d and 4-(diethylamino)salicylaldehyde 1e. To introduce the pyrimidine fragment on the 4H-chromene skeleton of 4, we transformed the 2-amino function of 4 into imidate 5. After addition of ethyl orthoformate (6 equivalents) to 4 in the presence of glacial acetic acid 0.5% mol. as catalyst, the resulting mixture was submitted to microwave dielectric heating at 110˚C - 140˚C for 60 min. After cooling down to room temperature, we observed that imidate 5 is not soluble in the crude reaction mixture which facilitates recovery by a simple filtration. Due to the success of this step for heating under microwave irradiation, we investigated access to 4-imino-4,5-dihydro-3H-chromeno[2,3-d]pyrimidin-3-amine 6 by aza-annulation of imidate 5 with hydrazine. Initial attempts after a set of experiments under microwave irradiation showed that the desired compound 6 was progressively decomposed, even we used low irradiation temperature (50˚C - 60˚C) and short irradiation time (30 - 45 min.). Applying a method described in literature [14] with slight modifications, treatment of imidate 5 with 6 equivalents of aqueous solution of hydrazine at room temperature in absolute ethanol afforded compound 6 as precipitate after only 15 min. The desired compound 6 was finally collected by simple filtration. According to this simple protocol, the compounds 6(a-e) were synthesized in yields (Table 1) ranging from 59% to 90%.
Scheme 1. Route used for the preparation of ethyl N-(3-cyano-4H-chromen-2-yl)formimidate derivatives 5(a-e) and 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine 6(a-e).
Table 1. Results for the preparation of ethyl N-(3-cyano-4H-chromen-2-yl)formimidate derivatives 5(a-e) and 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine 7(a-e).
Compound |
R |
React. Time(min.) |
React. Temp.(˚C) |
Yield (%)a |
Overall yield (%)b |
d of CH2 (ppm) |
5a |
8-MeO |
60 |
110 |
73 |
54 |
3.66 |
5b |
7-MeO |
60 |
140 |
80 |
59 |
3.59 |
5c |
5,6-(CH=CH)2 |
60 |
130 |
80 |
69 |
3.97 |
5d |
6-MeO |
60 |
140 |
70 |
37 |
3.65 |
5e |
7-Et2N |
60 |
110 |
62 |
26 |
3.58 |
6a |
8-MeO |
15 |
25 |
70 |
38 |
3.63 |
6b |
7-MeO |
15 |
25 |
81 |
48 |
3.57 |
6c |
5,6-(CH=CH)2 |
15 |
25 |
90 |
62 |
3.93 |
6d |
6-MeO |
15 |
25 |
70 |
26 |
3.63 |
6e |
7-Et2N |
15 |
25 |
59 |
15 |
3.31 |
aIsolated yield; bOverall yield calculated from compound 3.
The structure assignment of the new compounds 6(a-e) was evidenced by FTIR (Fourier-transform infrared spectroscopy) with NH band (νNH 3190 cm−1), NH2 band (3000 < ν < 3400 cm−1); by 1H NMR in DMSO-d6 (see Table 1 for CH2 signal of compounds 5 and 6) and also 13C NMR. In mass spectrometry analysis (HRMS), the [M+H]+ molecular ion signals for all compounds 6(a-e) were readily obtained as base signals.
2.2. Antitumor Evaluations of Compounds 4(a-e) and 6(a-e)
Table 2. Results for antiproliferative activity of the 2-amino-4H-chromene-3-carbonitrile 4(a-e) and the 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine 7(a-e).
Compounds |
% of survivala and IC50 (mM) of selected compoundsb |
4 and 6 |
R |
Huh7D12 |
Caco2 |
MDA- MB231 |
HCT116 |
PC3 |
MDAMB468 |
MCF7 |
Fibro- blast |
4a |
8-MeO |
100 |
89 |
104 |
94 |
95 |
102 |
106 |
NT |
4b |
7-MeO |
96 |
84 |
92 |
82 |
93 |
98 |
96 |
NT |
4c |
5,6-(CH=CH)2 |
95 |
88 |
103 |
81 |
98 |
94 |
97 |
NT |
4d |
6-MeO |
103 |
88 |
100 |
75 |
92 |
89 |
96 |
NT |
4e |
7-Et2N |
87 |
93 |
90 |
76 |
97 |
85 |
89 |
NT |
6a |
8-MeO |
89 |
87 |
88 |
85 |
97 |
96 |
91 |
NT |
6b |
7-MeO |
100 |
92 |
79 |
77 |
95 |
95 |
76 |
NT |
6c |
5,6-(CH=CH)2 |
8 (3) |
49 (3) |
17 (3) |
2 (2.1) |
27 (7) |
11 (1.8) |
31 (6) |
(>25) |
6d |
6-MeO |
89 |
95 |
97 |
67 (>25) |
86 |
88 |
89 |
(>25) |
6e |
7-Et2N |
81 |
104 |
85 |
61 (>25) |
83 |
62 (>25) |
78 |
(>25) |
DMSO |
100 (>25) |
100 (>25) |
100 (>25) |
100 (>25) |
100 (>25) |
100 (>25) |
100 (>25) |
(0.00) |
Roscovitinec |
12 (15) |
21 (13) |
23 (14) |
8 (14) |
27 (9) |
8 (8) |
20 (9) |
(13) |
Doxorubicinec |
61 (0.06) |
64 (0.03) |
37 (0.02) |
22 (0.03) |
41 (0.07) |
26 (0.07) |
37 (0.09) |
(0.01) |
Taxolc |
44 |
62 |
32 |
7 |
35 |
22 |
23 |
NT |
aPercentage of survival measured at 25 mM (after a single dose, triplicate); bIC50 values in brackets are expressed in mM and are the average of three assays, standard error ±0.5 Mm; cUsed as positive control.
Based on the antitumor activities reported in literature of bioactive compounds incorporating chrome or chromeno-pyrimidine moieties [1] [15]-[17], the 2-amino-4H-chromene intermediates 4(a-e) and their 4H-chromeno[2,3-d]pyrimidin derivatives 6(a-e) were evaluated on a panel of seven tumoral cell lines and diploid skin fibroblasts as normal cell lines for control. The roscovitin [18], doxorubicin [19] and taxol [20] compounds were used in all experiments as reference standards because they are wide-spectrum anticancer agents. For these in vitro antitumor assays, the used cell lines represented commons forms of human cancers, i.e., hepatocellular carcinoma (Huh7 D12), colon adenocarcinoma (Caco2), breast carcinoma (MDA-MB231), colon carcinoma (HCT116), triple negative breast cancer (MDA-MB468), breast cancer (MCF7), prostate adenocarcinoma (PC3).
The protocol for evaluation of antiproliferative activity takes place in two phases. The first phase is an evaluation of the target compound at a 25 μM single dose. When compounds exhibited a strong inhibitory activity with a percentage of survival is < 50% for the tumoral cell, the compound was used for a more detailed study in the second phase. Using different concentrations (0.1 - 25 μM), a study of full dose-response and survival curves was realized for determination of IC50 (concentration of the compound that kills 50% of the tumoral cell after 48 h incubation). Results for cytotoxic assays of compounds 4(a-e) and 6(a-e) are reported in Table 2. Analysis of the results in this table showed that most of the compounds 4 and 6 were inactive on the seven tumoral cell lines (percentage of survival is > 50% in phase 1 or IC50 > 25 μM in phase 2). The most interesting compound is 6c because IC50 < 10 μM on the seven tumoral cell lines and does not present toxic effect on fibroblasts. Indeed, compound 6c has interesting cytotoxic activities on Huh7 D12, Caco2 and MDA-MB231 with IC50 3 μM, on HCT116 (IC50 2.1 μM), on PC3 (IC50 7 μM), on MDA-MB468 (IC50 1.8 μM) and MCF7 (IC50 6 μM). It is noteworthy that this compound 6c does not present toxic effects on fibroblasts. In terms of cytotoxicity efficiency, we can notice that 6c is better than roscovitine and taxol on the seven tumoral cell lines but significantly less than doxorubicin. However, the latter is toxic towards fibroblasts.
2.3. In silico Physicochemical, ADME and Drug-Likeness Properties of Compound 6c
For the in silico physicochemicals properties of the bioactive compound 6c and also for the ADME pharmacokinetic properties, we used respectively the SwissADME [21] server platform [22] and the pkCSM web server [23]. The drug-likeness of compound 6c based on Lipinski’s rule of five (Ro5) [24] [25] was also predicted with the SwissADME web server.
In drug development, effective binding to the target is not only essential but also ensures oral bioavailability and drug-likeness properties based on Lipinski’s rule of five (Ro5) which includes molecular weight (MW), the octanol-water partition coefficient (log Po/w), the number of hydrogen bond acceptors (HBA), the number of hydrogen bond donors (HBD) and the topological polar surface area (tPSA). We find that these values are within the recommended range (Table 3) and it can be concluded that 6c had drug-ability characteristics according to Lipinski’s rules (violation = 0). Compound 6c presents favorable bioavailability score (0.55) that means prediction of good suitability for oral drug applications [26].
Table 3. In silico physicochemical properties and drug-likeness properties of compound 6c.
Parameters |
Compound 6c |
Range |
Molecular weight (MW) (g/mol) |
264.28 |
180 < MW < 500 |
# Heavy atoms |
20 |
20 < atoms > 70 |
# Aromatic heavy atoms |
16 |
- |
Fraction Csp3 |
0.07 |
- |
# Rotatable bonds (RB) |
0 |
<15 |
# H-bond acceptors (HBA) |
3 |
<10 |
# H-bond donors (HBD) |
2 |
<5 |
Molar refractivity (MR) |
75.93 |
40 < MR < 130 |
Topological Polar Surface Area, tPSA (Å2) |
76.92 |
tPSA < 140 Å2 |
Lipophilicity Log Po/w |
1.93 |
−0.4 < Log Po/w < +5.6 |
Water solubility Log S (Ali), class |
Soluble |
- |
Drug likeness (Lipinski Ro5), # violations |
Yes, 0 |
- |
Bioavailability Score |
0.55 |
- |
Lead-likeness, # violations |
Yes, 0 |
- |
Synthetic accessibility |
3.10 |
from 0 to 10, 10 is very difficult |
Csp3 = Fraction of carbon atoms in the sp3 hybridization; H = Hydrogen, # = number.
Predictive ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analyses offer valuable insights into have compound believe in the human body (Table 4 and Table 5). Compound 6c is predicted to have a good Caco2-permeability (1.248). The compound potential for distribution within the body was evaluated such as steady-state volume of distribution (VDss); in our case, 6c has VDss of 0.22. P-glycoprotein (P-gp) acts as a biological barrier [27], limiting the absorption of a compound from the gut by pumping xenobiotics out of cells to protect against toxic substances. Compound 6c is a substrate of P-gp and not a P-gp I/II inhibitor. Another aspect of 6c is their lower ability to traverse the blood-brain barrier (BBB), as evidenced by negative log BB (−0.155) but it can be distributed into the central nervous system, as evidenced by log PS value > −2 (log PS −2.152), which may have a potential neurological effect.
Table 4. ADME pharmacokinetic properties of compound 6c.
Parameters |
Compound 6c |
Range |
Absorption |
Water solubility (log mol/L) |
−2.78 |
Class of solubility: soluble < −2 |
Caco-2 permeability (log Papp in 10−6 cm/s) |
1.248 |
high Caco-2 permeability > 0.90 |
GI absorption |
95.82 |
poorly absorption when < 30% |
Skin permeability log Kp (cm/s) |
−2.76 |
low skin permeability if log Kp < −2.5 |
P-gp substrate (Yes/No) |
Yes |
- |
P-gp I inhibitor (Yes/No) |
No |
- |
P-gp II inhibitor (Yes/No) |
No |
- |
Distribution |
VDss (human) (Log L/kg) |
0.22 |
low if log VDss < −0.15, high if log VDss < 0.45 |
Fraction unbound (human) |
0.129 |
- |
BBB permeant |
−0.155 |
if log BB > 0.3 readily cross the BBB,if log BB < −1 poorly |
CNS permeability (log PS) |
−2.152 |
if log PS > −2 penetrate the CNS;if log < −3 unable |
Metabolism |
CYP2D6 substrate |
No |
- |
CYP3A4 substrate |
Yes |
- |
CYP1A2 inhibitor |
Yes |
- |
CYP2C19 |
Yes |
- |
CYP2C9 inhibitor |
Yes |
- |
CYP2D6 inhibitor |
No |
- |
CYP3A4 inhibitor |
No |
- |
Excretion |
Total real clearance (log ml/min/kg) |
0.716 |
- |
Renal OCT2 substrate |
No |
- |
Table 5. Toxicity properties of compound 6c.
Parameters |
Compound 6c |
Range |
Max. recommended tolerated dose MRTD (human) (log mg/kg/day) |
−0.153 |
if MRTD < ou = 0.477 low toxicity, if MRTD > 0.477 high |
hERG I inhibitor |
No |
- |
hERG II inhibitor |
No |
- |
Oral rat acute toxicity (LD50) (mol/kg) |
2.762 |
- |
Oral rat chronic toxicity (LOAEL) (log mg/kg/day) |
0.97 |
- |
AMES toxicity |
Yes |
- |
Hepatoxicity |
No |
- |
Skin sensitization |
No |
- |
After distribution, metabolism (Table 4) was conducted by assessing the interaction of compound 6c with cytochrome P450 isoforms, considering their role as substrate or inhibitor: 6c serve as substrate of CYP3A4, indicating possible modulation of metabolic pathways mediated by CYP3A4. Regarding excretion kinetics, compound 6c was not predicted as substrate of oral organic cation transporter 2 (OCT2) and exhibited a total real clearance of 0.716 log ml/min/kg, indicating its efficient elimination from the body.
The toxicity assessments outlined in Table 5 provide insights into the safety profiles of compound 6c. The value of oral rat acute toxicity (LD50 2.762 mol/kg) indicate immediate toxic effects and concerning the oral rat chronic toxicity (LOAEL 0.97 log mg/kg/day), the use of 6c suggests a potential long-term health risk associated with their usage, even at low dose of this potential cancer agent. On the contrary, 6c is not an inhibitor of hERG I/II and has no impact on cardiac health. Moreover, the absence of hepatotoxicity and skin sensitization are positive findings but 6c is predicted as potential mutagenic compound and may act as a carcinogen.
3. Conclusion
In conclusion, a new series of 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine 6(a-e) were synthesized in 5 steps. It was possible to prepare the imidates 5(a-e) under microwave dielectric heating. The structure of imidates 5(a-e) and new 4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine 6(a-e) was characterized on the basis of their infrared (FTIR), NMR spectral data. The 2-amino-4H-chromene intermediates 4(a-e) and their 4H-chromeno[2,3-d]pyrimidin derivatives 6(a-e) were evaluated on a panel of seven tumoral cell lines representing commons forms of human cancers and compared to diploid skin fibroblasts as normal cell lines for control. According to the results obtained, compounds 4(a-e), 6(a-b) and 6(d,e) exhibited low cytotoxic effects on tumoral cell lines compared to the three references (roscovitine, doxorubicine and taxol). Only the 4-imino-4H-benzo[5,6]-chromeno[2,3-d]pyrimidin-3(5H)-amine 6c showed interesting results on the 7 tumoral cell lines (1.8 < IC50 < 6 μm). This can be attributed to the dihydronaphto group. The predicted in silico ADMET profile of this compound 6c was in line with Lipinski’s rule of five (Ro5). This study will be an important guide for future in vivo studies and this structure can be considered interesting for further modification as very active anti-tumor agent.
4. Experimental Section
4.1. Chemistry Section
4.1.1. General Information
Thin-layer chromatography (TLC): on 0.2-mm precoated plates of silica gel 60 F-254 (Merck) with appropriate eluent. Visualization: with ultraviolet light (254 and 365 nm) or with a fluorescence indicator. Infrared (IR) spectra: recorded on a Jasco FT-IR 420 spectrophotometer apparatus using potassium bromide pellets. 1H NMR spectra: recorded on BRUKER AC 300 P (300 MHz) spectrometer. 13C NMR spectra: recorded on BRUKER AC 300 P (75 MHz) spectrometer. Chemical shifts: expressed in parts per million downfield. Data are given in the following order: d value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quint: quintuplet, m, multiplet; br, broad), number of protons, coupling constants J is given in Hertz. The high resolution mass spectra (HRMS): recorded in positive mode using direct Electrospray infusion, respectively on a Waters Q-Tof 2 or on a Thermo Fisher Scientific Q-Exactive spectrometers at the “Centre Régional de Mesures Physiques de l’Ouest” CRMPO (CRMPO platform, ScanMAT UAR 2025 CNRS, Rennes, France). Melting points: determined on a Kofler melting point apparatus and were uncorrected. Reactions under microwave irradiation (S2Wave platform ScanMAT UAR 2025 CNRS, Rennes): realized in the Anton Paar Monowave 300® microwave reactor (Anton Paar France) using borosilicate glass vials of 10 ml equipped with snap caps (at the end of the irradiation, cooling reaction was realized by compressed air). The microwave instrument consists of a continuous focused microwave power output from 0 to 800W. All the experiments were performed using stirring option. The target temperature was reached with a ramp of 3 minutes and the chosen microwave power stay constant to hold the mixture at this temperature. The reaction temperature is monitored using calibrated infrared sensor and the reaction time included the ramp period. The microwave irradiation parameters (power and temperature) were monitored by the Monowave software package for the Anton Paar Monowave 300® reactor.
4.1.2. General Procedure Under Microwave Irradiation for the Synthesis of Ethyl N-(3-Cyano-N-Substituted-4H-Chromen-2-yl)formimidate 5(a-e)
In a 20 ml glass tube were placed successively 2-amino-4H-chromen-3-carbonitrile 4 (2.5 mmol), commercial triethyl orthoformate (1.59 g, 15 mmol, 6 equiv) and glacial acetic acid (0.5% mol, 7 µl, 1.25 µmol). The glass tube was sealed with a snap cap and placed in the Microwave® 300 Anton Paar microwave cavity (P = 800 Watt). The reaction mixture under vigorous stirring (550 rpm) was irradiated at 110˚C -140˚C for 60 min. After microwave dielectric heating, the crude reaction mixture was allowed to cool down to room temperature. After standing, the insoluble compound 5 was collected by filtration on a Buchner funnel (porosity N˚ 4) and washed with Et2O (2 × 10 ml). Then the desired product 5 was dried under high vacuum at 25˚ (10−2 Torr) for 1 h to give a yellowish powder and was further used without purification.
Ethyl N-(3-cyano-8-methoxy-4H-chromen-2-yl)formimidate (5a):
Yield: 73%. Mp = 153˚C - 155˚C. IR (KBr, ν cm−1): 1674 (C=N), 2213 (C≡N). 1H NMR (DMSO-d6) δ 1.33 (t, 3H, J = 7.1 Hz, CH3); 3.64 (s, 2H, CH2); 3.81 (s, 3H, MeO); 4.33 (q, 2H, J = 7.1 Hz, =C-CH2); 6.73 (d, 1H, J = 7.6 Hz, H-7, Ar); 6.95 (d, 1H, J = 8.2 Hz, H-5, Ar); 7.07 (t, 1H, J = 7.9 Hz, H-6, Ar); 8.41 (s, 1H, CH).13C NMR (DMSO-d6) δ 14.3 (CH3, C-13); 25.7(CH2, C-4); 56.3 (CH3O, C-8’); 64.3 (CH2, C-12);74.3 (C-3, C=); 111.5 (C-7,Ar); 118.9 (CN); 119.1 (C-10,C=); 120.3 (C-5,Ar); 125.3 (C-6,Ar); 139.0 (C-9, C=); 148.0 (C-8, C=); 158.6 (C-2,C=); 160.9 (C-11). HRMS (ES+, MeOH/CH2Cl2: 9:1), m/z: 281. 0896 found (calculated for C14H14N2O3Na [M+Na]+ requires 281.08966).
Ethyl N-(3-cyano-7-methoxy-4H-chromen-2-yl)formimidate (5b):
Yield: 80%. Mp = 133˚C - 135˚C. IR (KBr, ν cm−1): 1689 (C=N), 2209 (C≡N). 1H NMR (DMSO-d6) δ 1.32 (t, 3H, J = 7.1 Hz, CH3); 3.59 (s, 2H, CH2); 3.74 (s, 3H, MeO); 4.32 (q, 2H, J = 7.1 Hz, =C-CH2); 6.68 (d, 1H, J = 2.5 Hz, H-8, Ar); 6.74 (dd, 1H, J = 8.4 Hz, J = 2.6 Hz, H-6, Ar); 7.11 (d, 1H, J = 8.4 Hz, H-5, Ar); 8.56 (s, 1H, CH).13C NMR (DMSO-d6) δ 14.3 (CH3, C-13); 25.0 (CH2, C-4); 55.9 (C-3, C=); 64.2 (CH2, C-12); 75.1 (CH3O, C-7’); 102.1 (C-5, Ar); 109.9 (C-6, Ar); 112.0 (C-10, Ar); 119.0 (CN); 129.9 (C-8, Ar); 150.3 (C-9, Ar); 158.4 (C-7, Ar); 159.5 (C-11); 161.4 (C-2, Ar). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 281.0898 found (calculated for C14H14N2O3Na [M+Na]+ requires 281.09021).
Ethyl N-(2-cyano-1H-benzo[f]chromen-3-yl)formimidate (5c):
Yield: 80%. Mp = 133˚C - 135 ˚C. IR (KBr, ν cm−1): 1674 (C=N), 2212 (C≡N). 1H NMR (DMSO-d6) δ 1.35 (t, 3H, J = 7.1 Hz, CH3); 3.97 (s, 2H, CH2); 4.35 (q, 2H, J = 7.0 Hz, =C-CH2); 7.29 (d, 1H, J = 8.9 Hz, H-8, Ar); 7.53 (t, 1H, J = 7.4 Hz, H-5’’, Ar); 7.63 (t, 1H, J = 7.6 Hz, H-6’’, Ar); 7.84 (d, 1H, J = 8.4 Hz, H-7, Ar); 7.90 (d, 1H, J = 9Hz, H-6’, Ar); 7.94 (d, 1H, J = 7.7 Hz, H-5’, Ar); 8.63 (s, 1H, CH). 13C NMR (DMSO-d6) δ 14.3 (CH3, C-13); 23.8 (CH2, C-4); 64.3 (C-3, C=); 75.0 (CH2, C-12); 111.0 (C-8, Ar); 117.5 (C-10, Ar); 119.1 (CN); 123.5 (C-5”, Ar); 125.8 (C-6’’, Ar); 127.8 (C-7, Ar); 128.7 (C-6’, Ar); 129.4 (C-5’, Ar); 131.0 (C-5, Ar); 131.1 (C-6, Ar); 146.8 (C-9, Ar); 158.2 (C-2, Ar); 161.6 (C-11). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 301.0949 found (calculated for C17H14N2O2Na [M+Na]+ requires 301.0953).
Ethyl N-(3-cyano-6-methoxy-4H-chromen-2-yl)formimidate (5d):
Yield: 70%. Mp = 197˚C - 199˚C; IR (KBr, ν cm−1): 1648 (C=N), 2207 (C≡N). 1H NMR (DMSO-d6) δ 1.32 (t, 3H, J = 7.1 Hz, CH3); 3.65 (s, 2H, CH2); 3.75 (s, 3H, MeO); 4.32 (q, 2H, J = 7.0 Hz, =C-CH2); 6.77 (s, 1H, H-5, Ar); 6.82 (d, 1H, J = 8.9 Hz, H-7, Ar); 7.02 (d, 1H, J = 8.9 Hz, H-8, Ar); 8.54 (s, 1H, CH). 13C NMR (DMSO-d6) δ 14.3 (CH3, C-13); 26.0 (CH2, C-4); 56.0 (C-3, C=); 64.2 (CH2, C-12); 73.8 (CH3O, C-6’); 113.3 (C-5, Ar); 114.5 (C-10, Ar); 117.9 (C-7, Ar); 119.1 (CN); 119.2 (C-8, Ar); 143.6 (C-9, Ar); 156.6 (C-6, Ar); 158.8 (C-2, Ar); 161.4 (C-11). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 281.0901 found (calculated for C14H14N2O3Na [M+Na]+ requires 281.0902).
Ethyl N-(3-cyano-7-(diethylamino)-4H-chromen-2-yl)formimidate (5e):
Yield: 62%. Mp = 110˚C - 112˚C. IR (KBr, ν cm−1): 1650 (C=N), 2213 (C≡N). 1H NMR (CDCl3) δ 1.18 (t, 6H, J = 7.0 Hz, 2-CH3); 1.41 (t, 3H, J = 7.1 Hz, CH3); 3.34 (q, 4H, J = 7.0 Hz, 2-CH2-N); 3.58 (s, 2H, CH2); 4.44 (q, 2H, J = 7.1 Hz, CH2CH3); 6.24 (d, 1H, J = 2.1 Hz, H-8, Ar); 6.46 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz, H-6, Ar); 6.93 (d, 1H, J = 8.5 Hz, H-5, Ar); 8.35 (s, 1H, CH).13C NMR (DMSO-d6) δ 12.5 (CH3, C-7c, C-7d); 13.9 (CH3, C-13); 44.4 (C-7a, C-7b, CH2); 63.8 (CH2CH3, C-12); 75.6 (C-3); 99.1 (C-8, Ar); 103.4 (C-10, Ar); 109.0 (C-6, Ar); 119.2 (CN); 129.0 (C-5, Ar); 148.0 (C-9, Ar); 150.6 (C-7, Ar); 158.3 (C-11); 158.6 (C-2, Ar). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 355.1169 found (calculated for C19H16N4O2Na [M+Na]+ requires calculated for 355.11710).
4.1.3. General Procedure for the Preparation of 4-Imino-4H-Chromeno[2,3-d]pyrimidin-3(5H)-Amine 6(a-e)
To a vigorous stirred solution (550 rpm) of ethyl N-(3-cyano-4H-chromen-2-yl) formimidate 5 (5 mmol) in absolute ethanol (20 ml) was added portion wise hydrazine monohydrate (1.5 g, 30 mmol, 6 equiv.). After 3 min., a precipitate appeared and mixing was pursued at 25˚C until complete precipitation (15 min.). The insoluble material was collected by filtration in a Büchner funnel (porosity N˚4) and the solid product was washed with cooled ethanol (2 × 10 ml) to give the desired product 6. Drying under high vacuum at 25˚ (10−2 Torr) for 1h produced 6 as a yellowish powder which was recrystallized from ethanol.
4-Imino-9-methoxy-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine (6a):
Yield: 70%, Mp > 250˚C. IR (KBr, ν cm−1): 1648 (C=N), 3212 (NH), 3296/3331 (NH2). 1H NMR (DMSO-d6) δ 3.47 (s, 1H, NH); 3.63 (s, 2H, CH2); 3.81 (s, 3H, MeO); 5.70 (s, 2H, NH2); 6.79 (d, 1H, J = 7.4 Hz, H-8, Ar); 6.92 (d, 1H, J = 7.8 Hz, H-6, Ar); 7.03 (t, 1H, J = 7.9 Hz, H-7, Ar); 8.01 (s, 1H, CH). 13C NMR (DMSO-d6) δ 23.7 (CH2, C-5); 56.2 (CH3, C-10); 94.9 (C-4a, C=); 111.2 (C-8, Ar); 120.2 (C-5a, Ar); 120.9 (C-6, Ar); 120.5 (C-7, Ar); 139.7 (C-9a, Ar); 148.1 (C-4, Ar); 150.4 (C-2, Ar); 158.9 (C-9, Ar). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 245.1038 found (calculated for C12H13N4O2 [M+H]+ requires 245.1039).
4-Imino-8-methoxy-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine (6b):
Yield: 81%. Mp ≥ 260˚C. IR (KBr, ν cm−1): 1654 (C=N), 3208/3212 (NH/NH2). 1H NMR (DMSO-d6) δ 3.57 (s, 2H, CH2); 3.74 (s, 3H, MeO); 5.70 (s, 2H, NH2); 6.64 (s, 1H, H-9, Ar); 6.70 (d, 1H, J = 6.4 Hz, H-7, Ar); 7.15 (d, 1H, J = 8.4 Hz, H-6, Ar); 8.02 (s, 1H, CH). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 245.1034 found (calculated for C12H13N4O2 [M+H]+ requires 245.1039).
4-Imino-4H-benzo[5,6]-chromeno[2,3-d]pyrimidin-3(5H)-amine (6c):
Yield: 90%. Mp ≥ 260˚C. IR (KBr, ν cm−1): 1650 (C=N), 3179 (NH), 3281/3301 (NH2). 1H NMR (DMSO-d6) δ 3.93 (s, 2H, CH2); 5.78 (s, 2H, NH2); 7.30 (d, 1H, J = 8.9 Hz, H-9); 7.53 (t, 1H, J = 7.1 Hz, H-6’’, Ar); 7.66 (t, 1H, J = 8.1Hz, H-7’’, Ar); 7.88 (d, 1H, J = 8.9 Hz, H-8, Ar); 7.95 (d, 1H, J = 8.0 Hz, H-7’, Ar); 8.01 (d, 1H, J = 8.4Hz, H-6’, Ar); 8.10 (s, 1H, CH). 13C NMR (DMSO-d6) δ 21.8 (CH2, C-5); 95.0 (C-4a, C=); 111.9 (C-9, Ar); 117.7 (C-5a, C=); 123.7 (C-8, Ar); 125.4 (C-6’, Ar); 127.6 (C-7’, Ar); 128.7 (C-6’’, Ar); 129.1 (C-7’’, Ar); 130.7 (C-6, C=); 131.8 (C-7, C=); 147.4 (C-2, Ar); 150.4 (C-4, C=); 155.6 (C-9a, C=). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 265.1085 found (calculated for C15H13N4O [M+H]+ requires 265.10894).
4-Imino-7-methoxy-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine (6d):
Yield: 70%. Mp ≥ 260˚C. IR (KBr, ν cm−1): 1650(C=N), 3179(NH), 3281/3301 (NH2). 1H NMR (DMSO-d6) δ 3.63 (s, 2H, CH2); 3.73 (s, 3H, MeO); 5.69 (s, 2H, NH2); 6.81-6.78 (m, 2H, H-8, H-6, Ar); 7.00 (d, 1H, J = 9.6 Hz, H-9, Ar); 8.01 (s, 1H, CH). 13C NMR (DMSO-d6) δ 24.0 (CH2, C-5); 55.8 (CH3, C-7a); 94.3 (C-4a, C=); 113.9 (C-8, Ar); 114.1 (C-6, Ar); 117.7 (C-9, Ar); 120.3 (C-5a, C=); 144.1 (C-9a, C=); 150.5 (C-4, C=); 156.1 (C-2, Ar). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 245.1031 found (calculated for C12H13N4O2 [M+H]+ requires 245.10385).
N-diethylamino-4-imino-4H-chromeno[2,3-d]pyrimidin-3(5H)-amine (6e):
Yield: 59%. Mp 212˚C; IR (KBr, ν cm−1): 1645 (C=N), 3250 (NH), 3279/3320 (NH2). 1H NMR (DMSO-d6) δ 1.07 (t, 6H, 2-CH3); 3.33 (q, 6H, 3-CH2); 3.49 (s, 1H, NH); 5.69 (s, 2H,NH2); 6.26 (s, 1H, H-9, Ar); 6.45 (d, 1H, 3J = 6 Hz, H-7, Ar); 7.01 (d, 1H, J = 6 Hz, H-6, Ar); 8.01 (s, 1H, CH).13C NMR (DMSO-d6) δ 12.8 (CH3, C-8’’’, C-8’’); 22.7 (CH2, C-5); 44.2 (CH2, C-8’, C-8’’); 95.4 (C-4a, C=); 99.2 (C-9, Ar); 105.1 (C-5a, C=); 108.9 (C-7, Ar); 130.1 (C-6, Ar); 147.9 (C-2, Ar); 150.1 (C-4, C=); 151.1 (C-9a, C=); 156.2 (C-8, C=). HRMS (ES+, MeOH/CH2Cl2 9:1), m/z: 268.1658 found (calculated for C15H20N5O [M+H]+ requires 286.16679).
4.2. Biology Section for Antiproliferative Assays
4.2.1. Cell Culture
Skin diploid fibroblastic cells were provided by BIOPREDIC International Company (Rennes, France). Huh-7D12 (Ref ECACC: 01042712), Caco2 (Ref ECACC: 86010202), MDA-MB-231 (Ref ECACC: 92020424), MDA-MB-468, HCT-116 (Ref ECACC: 91091005), PC3 (Ref ECACC: 90112714), and MCF7 cell lines were obtained from the ECACC collection. Cells were grown according to ECACC recommendations [28] in DMEM for Huh-D12, MDA-MB-231, MDA-MB-468 and fibroblast, in EMEM for Caco2, in McCoy’s for HCT-116 and RPMI for PC3 at 37˚C and 5% CO2. All culture media with 10% of FBS, 1% of penicillin-streptomycin and 2 mM glutamine.
4.2.2. Protocol for Antiproliferative Assays
Chemicals were solubilized in DMSO at a concentration of 10 mM (stock solution) and diluted in culture medium to the desired final concentrations. The dose effect cytotoxic assays (IC50 determination) were performed by increasing concentrations of each chemical (final concentrations: 0.1 μM, 0.3 μM, 0.9 μM, 3 μM, 9 μM and 25 μM). The toxicity test of the chemicals on these cells was as follows: 2 × 103 cells for HCT-116 cells or 4 × 103 for the other cells were seeded in 96-multi-well plates in triplicate and left for 24 h for attachment, spreading and growing. Then, cells were exposed for 48 h to increasing concentrations of chemicals, ranging from 0.1 μM to 25 μM, in a final volume of 120 μL of culture medium. After 48 h of treatment, cells were washed in PBS and fixed in cooled 90% ethanol/5% acetic acid solution for 20 min. The nuclei were stained with Hoechst 3342 (B2261 Merck Sigma-Aldrich) and counted. Image acquisition and analysis were performed using automated imaging analysis with a Cellomics Arrayscan VTI/HCS Reader (Thermo/Scientific, Waltham, MA, USA). The survival percentages were calculated as the percentage of cell number after chemical treatment over cell number after DMSO treatment. The IC50 were graphically determined.
4.3. In Silico Pharmacokinetics
The SMILES of compound 6c was used for in silico ADME (absorption, distribution, metabolism, and excretion) screening on SwissADME server [22], which was performed at default parameters. Also, the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) study of 6c were calculated using the pkCSM server (http://biosig.unimelb.edu.au/pkcsm/, [23]).
Author Contributions
Organic synthesis, S.K., M.D. and M.F.; supervision for organic synthesis, H.A., S.A., L.P. and J-P.B.; hygiene and security training for organic synthesis, technical formation for microwave irradiation synthesis, E.L.; tumoral cell lines assays, R.L.G.; tumoral cell lines assays, R.L.G.; T.C., supervision of tumoral cell lines assays; in silico ADME prediction on SwissADME and pkCSM-pharmacokinetics servers, S.K. and J-P.B.; supervision of project, H.A. and J-P.B.; writing-article and editing, J-P.B., funding acquisition of Canceropôle Grand-Ouest contract, J-P.B.; funding acquisition for junior and senior mobility of Campus France, H.A. and J-P.B. All authors have read and agreed to the publication version of the manuscript.
Funding
This research work was funded by the “Ministère de l’Enseignement Supérieur et de la Recherche de la République Tunisienne’’ (PhD fellowship for S.K. and M.D.). The biological assays for this research program were supported by the “Canceropôle Grand-Ouest’’ in “Molecules Marines, Metabolisme et Cancer 3MC’’ network. This project has received funding for mobility of Tunisian junior and senior researchers (S.K., H.A.) and French senior researchers (L.P., E.L. and J-P.B.) by Campus France via PHC Utique 2021-23 contract (Campus France code 46156NL/CMCU code 21G1202) and also funding for mobility of M.D. by University of Sfax.
Acknowledgments
The authors are grateful to the assistance of the staff (N. Le Yondre, P. Jéhan, F. Lambert) of CMRPO analytical chemistry core facility for HRMS analysis (CRMPO platform UAR 2025 CNRS, Université de Rennes 1, Bât. 11A, Campus de Beaulieu, Rennes, France).