CN107536835A - Application and medicine of 1,3 diaminourea 7H pyrroles [3,2 f] quinazoline derivant as antibacterials - Google Patents

Abstract

The application of a 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivative as an antibacterial drug and an antibacterial drug containing the compound, the compound chemical structure general formula I is as follows: Y is selected from an alkane group with 1-5 carbon chains, Z is selected from -COOCH ₃ , -OCH ₃ , -CF ₃ , Cl, Br, F, R ₁ is selected from H, -CH ₃ , R ₂ is selected from H, -CH ₃ , wherein the bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis.

Description

1,3-Diamino-7H-pyrrole[3,2-f]quinazoline derivatives as antibacterial drugs Applications and Drugs

technical field

The invention relates to the field of medicine, in particular to the antibacterial medical application of 1,3-diamino-7H-pyrrole [3,2-f]quinazoline derivatives and antibacterial drugs containing the compounds.

Background technique

Dihydrofolate reductase (DHFR) can catalyze the reduction of dihydrofolate to tetrahydrofolate, and is the precursor of thymidylate for the subsequent synthesis of purine nucleotides, methionine, serine, and glycine. However, these compounds are the necessary raw materials for the DNA, RNA and proteins required for biological reproduction. Therefore, inhibiting the catalytic activity of dihydrofolate reductase (DHFR) can hinder the DNA replication process of cells. Currently reported DHFR inhibitors have a variety of biological activities, such as anticancer activity, antimalarial activity, anti-Mycobacterium tuberculosis activity, antibacterial activity and so on. From the perspective of chemical structure, DHFR inhibitors can be divided into two types: "classical" and "non-classical". Usually, classical DHFR inhibitors have a structure similar to folic acid, such as methotrexate; non-classical DHFR inhibitors do not have similar structures to folic acid, and their core structure is 2,4-diaminopyrimidine structure. Compared with classic DHFR inhibitors, non-classical DHFR inhibitors do not have a glutamic acid residue structure in their molecules. They are more fat-soluble and can enter cells through passive diffusion without the process of polyglutamic acid. Therefore, relatively speaking, non-classic DHFR inhibitors can reduce the degree of drug resistance of cells, reduce the toxic effect on normal human cells to a certain extent, and improve the effect of drugs. Such as quinazoline DHFR inhibitors Pelitrine, trimetrexate and so on. They are more effective in inhibiting DHFR, and have higher oral bioavailability, high clearance rate, and less impact on histamine metabolizing enzymes.

However, the structure of the above-mentioned classic DHFR inhibitors is still relatively complex, difficult to synthesize, and the cost of preparation is high, and the activity is not strong enough, especially the relatively simple structure, which is easy to produce new drug dependence.

Hong Wei, Wang Hao et al. (Hong, W. et al. The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modeling. Sci. Rep. 5, 15328; doi: 10.1038/srep15328, 2015) via computer Through virtual screening, 1,3-diamino-7H-pyrrole[3,2-f]quinazoline was found as the core nucleus of Mycobacterium tuberculosis DHFR inhibitors, and a series of 1,3-diamino-7H -Pyrrole[3,2-f]quinazoline derivatives (see Wang Hao, Hong Wei, Ouyang Yifan, Yang Hao, Tan Xiaoli, Wang Zhe, Sun Yutong, Zhu Xuanli, Chinese Patent CN106632350A, publication date 2017.05.10). However, follow-up activity tests showed that the antibacterial activity of this series of compounds was not ideal, especially against certain drug-resistant bacteria.

Contents of the invention

The present invention is carried out in order to solve the above-mentioned problem, has proposed the compound of 1,3-diamino-7H-pyrrole [3,2-f] quinazoline of another structure as core, and it has more than the analogous compound disclosed before Better antibacterial activity, especially the potential activity against multidrug-resistant Acinetobacter baumannii, is expected to further develop clinically available antibacterial drugs that can inhibit multidrug-resistant Acinetobacter baumannii.

A kind of 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivative is used as the application of antibacterial drug, and the general chemical structure formula I of this compound is as follows:

in,

Y is selected from alkane groups with 1-5 carbon chains,

Z is selected from -COOCH ₃ , -OCH ₃ , -CF ₃ , Cl, Br, F,

R ₁ is selected from H, -CH ₃ ,

R ₂ is selected from H, -CH ₃ .

The application of 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention as antibacterial drugs can also have such characteristics, characterized in that: wherein, Y is selected from 1- 3 carbon chain alkane groups, para-substituted

The application of 1,3-diamino-7H-pyrrole [3,2-f]quinazoline derivatives provided by the present invention as antibacterial drugs can also have such characteristics, characterized in that: wherein, Z is selected from para Substituted -COOCH ₃ , para-substituted -OCH ₃ , para-substituted -CF ₃ , ortho or para or meta substituted Cl, Br, F.

The application of 1,3-diamino-7H-pyrrole [3,2-f]quinazoline derivatives provided by the present invention as antibacterial drugs can also have such characteristics, characterized in that: wherein, R ₁ , R ₂ Both are H.

The 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention are used as antibacterial drugs, and can also have such characteristics, characterized in that they are selected from compounds of the following structures:

Among them, bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis.

The present invention also provides a medicament for antibacterial application containing 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives, the general chemical structure formula I of the compound is as follows:

in,

Y is selected from alkane groups with 1-5 carbon chains,

Z is selected from -COOCH ₃ , -OCH ₃ , -CF ₃ , Cl, Br, F,

R ₁ is selected from H, -CH ₃ ,

R ₂ is selected from H, -CH ₃ .

The antibacterial drug containing 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention can also have such a feature, wherein, Y is selected from 1 - 3 carbon chain alkane groups, para-substituted

The antibacterial drug containing 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention can also have such a feature, wherein, Z is selected from the group consisting of Position substituted -COOCH ₃ , para position substituted -OCH ₃ , para position substituted -CF ₃ , ortho or para or meta substituted Cl, Br, F.

The antibacterial drug containing 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention can also have such characteristics, characterized in that: wherein, R ₁ , R ₂ are H.

The antibacterial drug containing 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the present invention can also have such a feature, characterized in that it is selected from compounds with the following structures :

Among them, bacteria include Klebsiella pneumoniae ATCC700603 producing extended-spectrum β-lactamase, multidrug-resistant Acinetobacter baumannii ATCC19606, methicillin-resistant Staphylococcus aureus ATCC43300, and vancomycin-resistant Enterococcus faecium ATCC51299, Escherichia coli ATCC25922.

Invention function and effect

The preparation method of 1,3-diamino-7H-pyrrole [3,2-f] quinazoline derivatives provided by the invention adopts the new raw material II and _BF3 to react, and the post-reaction treatment is convenient, by simple After the alkalization treatment, the precipitated product can be separated by filtration, and the yield is high.

Because the preparation method provided by the invention is a one-pot method, the intermediate does not need to be purified; when compound III is converted into compound II, the method in the literature is to first salt indole and then react with sodium dicyanamide. The organic acid p-toluenesulfonic acid is used as a catalyst to directly react with sodium dicyanamide, and the method is simplified.

Further, the obtained 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j were tested for antibacterial activity in vitro, and it was concluded that:

1. The compound has extremely low-low MIC against Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) Value (0.0004μg/mL-32μg/mL), reflecting a strong broad-spectrum antibacterial activity. Among them, the MIC values of 6a, 6b, 6c, 6d, 6e, 6f, and 6g against Staphylococcus aureus ATCC29213 were ≤0.06 μg/mL; the MIC values for MRSA ATCC43300 ranged from 0.06 μg/mL to 0.25 μg/mL; against Escherichia coli ATCC25922 The range of MIC value is 0.0004μg/mL-0.06μg/mL; the range of MIC value for broad-spectrum β-lactamase-producing Klebsiella pneumoniae is 0.25μg/mL-2μg/mL; The MIC of Enterococcus ATCC51299≤0.06μg/mL. The MICs of 6d and 6e against multidrug-resistant Acinetobacter baumannii ATCC19606 were 0.006μg/mL and 0.125μg/mL, respectively. It is comprehensively shown that this type of compound has extremely low MIC value against Gram-negative bacteria, especially multidrug-resistant Acinetobacter baumannii, and shows strong antibacterial activity.

2. Compounds 6d, 6e, 6f, and 6i have MIC50 and MIC90 ranges of 8 μg/mL-16 μg/mL for 41 strains of clinically isolated multidrug-resistant Acinetobacter baumannii from 2006 to 2010, which are superior to most existing antibacterial agents Drugs, and there is no cross-resistance with existing antibacterial drugs such as carbatherene.

3. By testing the bactericidal curves of compound 6d on Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922, it was found that this compound has different bactericidal characteristics on G+ bacteria and G- bacteria.

4. The bactericidal curve of compound 6d on Escherichia coli ATCC25922 showed that different concentrations of compound 6d showed antibacterial effect at 0-8 hr, and the antibacterial effect had no obvious concentration dependence, and it could occur at a very low level of compound concentration ( About 2×10 ^-8 μg/mL), the maximum bactericidal effect is reached in 8～24hr. In the concentration range of 1/8×MIC～256×MIC, the bactericidal effect can be maintained for at least 48 hours.

5. The bactericidal curve of compound 6d on Staphylococcus aureus ATCC29213 shows that this compound has a bactericidal effect on Staphylococcus aureus ATCC29213, and the bactericidal effect is rapid in 4 to 8 hours, and the bactericidal effect is between 1/4×MIC and 4×MIC concentration Can keep at least 48hr.

The above activity tests show that the compound provided by the invention has a good inhibitory effect on various clinically infected bacteria, especially a good inhibitory effect on clinical multidrug-resistant bacteria, and it is proved by controlled experiments that this Compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, and 6j disclosed by the invention have better in vitro antibacterial activity than a variety of antibacterial drugs used in clinical frontline, and have better antimicrobial activity against a variety of drug-resistant bacteria. Antibacterial effect.

Description of drawings

Fig. 1 is the bactericidal curve of 6d to escherichia coli ATCC25922; And

Fig. 2 is the bactericidal curve of 6d to Staphylococcus aureus ATCC29213.

detailed description

In order to make the technical means realized by the present invention, creative features, goals and effects easy to understand, the following examples are for the preparation of 1,3-diamino-7H-pyrrole [3,2-f]quinazoline derivatives of the present invention The method and the physicochemical and spectral data of the compound are described in detail.

Compound route:

Experimental part:

Synthetic general method of compound 3a-j: Add 5-nitroindole (6.17mmol) into anhydrous DMSO (30ml), add 60% NaH (7.40mmol) in portions under the protection of argon, and stir at room temperature for 1h. Then compound 2a-j (7.40mmol) was added to the mixture, stirred at room temperature for 3h. The reaction was quenched by adding saturated ammonium chloride solution, extracted with ethyl acetate, the ester layer was washed with distilled water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product.

Synthetic general method of compound 4a-j: add compound 3a-j (4.28mmol), iron powder (21.41mmol) and ammonium chloride (42.82mmol) in ethanol-water (4:1) (50ml), reflux at 80°C The reaction was stirred for 4h. The reaction solution was rotary evaporated, distilled water was added to the residue to dissolve, the pH value was adjusted to alkaline with anhydrous sodium bicarbonate, extracted with dichloromethane, the organic layer was washed with distilled water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product.

Synthetic general method of compound 5a-j: Dissolve compound 4a-j (3.12mmol) in anhydrous DMF (10ml), then add p-toluenesulfonic acid (3.12mmol) and NaN(CN) ₂ (9.21mmol) , and the reaction was stirred overnight at 50°C. The reaction liquid was poured into water which was about 4 times of the solvent, stirred, suction filtered, and the filter cake was dried to obtain a crude product.

General synthesis of compound 6a-j: Compound 5a-j (2.58mmol) was added to DME (15ml), BF ₃ -Et ₂ O (12.91mmol) was added under ice-cooling conditions, and the reaction was stirred overnight at 60°C. Rotate the reaction solution, dissolve the residue in a small amount of methanol, add 1mol/L NaOH solution, stir, filter with suction, and dry the filter cake to obtain the crude product. The product was purified by column chromatography using dichloromethane:methanol=20:1 (V:V) as the eluent.

Compound 6a, 1,3-diamino-7-(4-methoxycarbonyl-benzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.82g; yield 92%. mp 173.6-174.8°C; IR 3503, 3458, 3316, 3148, 1703, 1632, 1577, 1556, 1518, 1476, 1358, 1289, 935, 818, 741cm ^-1 ; ¹ HNMR (400MHz, DMSO-d6) δ7. 93-7.87(m, 2H), 7.70(d, J=9.0Hz, 1H), 7.66(d, J=3.1Hz, 1H), 7.28-7.24(m, 2H), 7.15(d, J=3.1Hz , 1H), 7.03 (d, J=9.0Hz, 1H), 6.83 (s, 2H), 5.83 (s, 2H), 5.62 (s, 2H), 3.82 (s, 3H); ¹³ CNMR (101MHz, DMSO -d6) δ166.37, 162.41, 159.31, 149.74, 144.24, 130.71, 129.95, 129.37, 129.15, 127.45, 121.40, 119.36, 117.92, 102.99, 102.30, 52.58, 49.39; ⁺ , 100); HRMS (ESI) m/z [M+H] ⁺ Calcd for C ₁₉ H ₁₈ N ₅ O ₂ : 348.1460, Found: 348.1455.

Compound 6b, 1,3-diamino-7-(4-trifluoromethyl-benzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.85g; yield 92%. mp 143.8-144.8°C; IR 3326, 3171, 1583, 1519, 1495, 1358, 1326, 1111, 1066, 1017, 936, 819, 750, 716cm-1; 1H NMR (400MHz, DMSO-d6) δ7.76( d, J=9.0Hz, 1H), 7.72-7.66(m, 3H), 7.34(d, J=8.0Hz, 2H), 7.19(d, J=3.1Hz, 1H), 7.06(d, J=9.0 Hz, 1H), 6.99(s, 2H), 6.01(s, 2H), 5.65(s, 2H); 13C NMR (101MHz, DMSO-d6) δ162.51, 158.69, 148.31, 143.52, 130.89, 129.72, 127.90 , 126.00, 125.96, 121.45, 118.48, 118.15, 102.84, 102.33, 49.19; MS (ESI) m/z: 358.1 (M+, 100); HRMS (ESI) m/z [M+H]+Calcd forC18H15F3N5: 358.1280, Found: 358.1287.

Compound 6c, 1,3-diamino-7-(4-bromobenzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.85g; yield 90%. mp 224.1-225.4°C; IR 3425, 3074, 1588, 1519, 1490, 1466, 1439, 1360, 1294, 1274, 1071, 1040, 1011, 938, 817, 798, 758, 718cm ^-1 ; ¹ H NMR (400MHz , DMSO-d6) δ7.72 (d, J = 9.0Hz, 1H), 7.63 (d, J = 3.1Hz, 1H), 7.54-7.48 (m, 2H), 7.15-7.10 (m, 3H), 7.03 (d, J=8.9Hz, 1H), 6.79(s, 2H), 5.80(s, 2H), 5.49(s, 2H); ¹³ C NMR (101MHz, DMSO-d6) δ162.38, 159.33, 138.19, 131.92, 130.60, 129.54, 129.22, 121.40, 120.94, 119.37, 117.95, 102.98, 102.21, 49.04; MS (ESI) m/z: 368.1 (M ⁺ , 100); HRMS (ESI) m/z [M+H] ⁺ Calcd for C ₁₇ H ₁₅ BrN ₅ : 368.0511, Found: 368.0510.

Compound 6d, 1,3-diamino-7-(4-fluorobenzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.75g; yield 95%. mp 179.8-181.2°C; IR 3320, 3167, 1620, 1581, 1555, 1510, 1442, 1357, 1272, 1222, 1157, 1067, 936, 820, 725cm ^-1 ; 1H NMR (400MHz, DMSO-d6) δ7. 79(d, J=9.0Hz, 1H), 7.67(d, J=3.1Hz, 1H), 7.29-7.23(m, 2H), 7.19-7.11(m, 3H), 7.05(d, J=8.9Hz , 1H), 6.93(s, 2H), 5.96(s, 2H), 5.50(s, 2H); ¹³ C NMR (101MHz, DMSO-d6) δ162.49, 158.80, 148.56, 134.88, 134.85, 130.76, 129.53 , 129.45, 121.40, 118.51, 118.20, 115.92, 115.71, 102.84, 102.10, 48.97; MS (ESI) m/z: 308.1 (M ⁺ , 100); HRMS (ESI) m/z [M + H] ⁺ Calcdfor C ₁₇ H ₁₅ FN ₅ : 308.1311, Found: 308.1309.

Compound 6e, 1,3-diamino-7-(3-fluorobenzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.73g; yield 92%. mp 229.9-231.6°C; IR 3425, 3076, 1620, 1588, 1519, 1494, 1463, 1435, 1357, 1279, 1246, 1189, 1132, 1074, 937, 818, 780, 715cm ^-1 ; ¹ H NMR (400MHz , DMSO-d6) δ7.77(d, J=9.0Hz, 1H), 7.67(d, J=3.1Hz, 1H), 7.35(td, J=7.7, 5.9Hz, 1H), 7.15(d, J =3.1Hz, 1H), 7.12-6.98(m, 4H), 6.86(s, 2H), 5.88(s, 2H), 5.54(s, 2H); 13C NMR (101MHz, DMSO-d6) δ163.88, MS( ^ESI )m/z: 308.1( ESI) m/z [M+H] ⁺ Calcd for C ₁₇ H ₁₅ FN ₅ : 308.1311, Found: 308.1311.

Compound 6f, 1,3-diamino-7-(2-fluorobenzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.70g; yield 88%. mp 193.0-194.6°C; IR 3428, 3082, 1620, 1584, 1519, 1489, 1357, 1272, 1037, 934, 835, 817, 754, 703cm ^-1 ; ¹ H NMR (400MHz, DMSO-d6) δ7.81 (d, J=9.0Hz, 1H), 7.62 (d, J=3.1Hz, 1H), 7.33 (tdd, J=7.7, 5.5, 2.7Hz, 1H), 7.24 (ddd, J=10.5, 8.3, 1.2 Hz, 1H), 7.17(d, J=3.2Hz, 1H), 7.14-7.06(m, 2H), 6.99(td, J=7.7, 1.9Hz, 3H), 6.04(s, 2H), 5.58(s , 2H); ¹³ C NMR (101MHz, DMSO-d6) δ162.54, 158.60, 148.09, 130.97, 130.26, 129.74, 129.64, 125.44, 125.30, 125.15, 121.28, 118.18, 116.01, 115.803, 102.6; MS (ESI) m/z: 308.1 (M ⁺ , 100); HRMS (ESI) m/z [M+H] ⁺ Calcd for _C17H15FN5 : _308.1311 , Found: _308.1317 .

Compound 6g, 1,3-diamino-7-(4-methoxy-benzyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.76g; yield 92%. mp 184.3-185.5°C; IR 3490, 3338, 3197, 1661, 1583, 1549, 1516, 1494, 1358, 1253, 1177, 1031, 936, 816, 763, 702cm ^-1 ; ¹ H NMR (400MHz, DMSO-d6 )δ7.87(d, J=9.0Hz, 1H), 7.71(d, J=3.1Hz, 1H), 7.26(s, 2H), 7.21-7.16(m, 3H), 7.09(d, J=8.9 Hz, 1H), 6.92-6.84(m, 2H), 6.32(s, 2H), 5.44(s, 2H), 3.70(s, 3H); ¹³ C NMR (101MHz, DMSO-d6) δ162.71, 159.08 , 157.50, 145.55, 131.20, 130.36, 130.12, 128.93, 121.39, 118.77, 116.43, 114.40, 102.52, 101.77, 55.52, 49.31; MS (ESI) m/z: 320.1 (M ⁺ , 100); HRMS (ESI /z[M+H] ⁺ Calcd for C ₁₈ H ₁₈ N ₅ O: 320.1511, Found: 320.1513.

Compound 6h, 1,3-diamino-7-(cyclopropylmethyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.59g; yield 90%. mp 200.0-201.8°C; IR 3479, 3384, 3124, 1653, 1625, 1578, 1554, 1519, 1494, 1459, 1361, 1264, 1244, 1118, 928, 818, 722cm ^-1 ; ¹ H NMR (400MHz, DMSO -d6) δ7.89(d, J=8.9Hz, 1H), 7.60(d, J=3.1Hz, 1H), 7.14-7.09(m, 2H), 7.07(s, 2H), 6.12(s, 2H ), 4.13(d, J=7.0Hz, 2H), 1.30-1.21(m, 1H), 0.55-0.49(m, 2H), 0.42-0.37(m, 2H); ¹³ C NMR (101MHz, DMSO-d6 )δ162.63, 158.11, 147.02, 131.14, 129.23, 121.04, 118.32, 117.22, 102.63, 101.43, 50.33, 12.31, 4.21; MS (ESI) m/z: 254.1 (M ⁺ , 100); HRMS (ESI) m /z[M+H] ⁺ Calcd for C ₁₄ H ₁₆ N ₅ : 254.1406, Found: 254.1402.

Compound 6i, 1,3-diamino-7-(thiophene-2-methyl)-7H-pyrrole[3,2-f]quinazoline

White solid 0.70 g; yield 92%. mp 184.5-185.6°C; IR 3435, 3311, 3127, 1617, 1584, 1515, 1489, 1453, 1431, 1356, 1239, 1187, 1120, 1036, 933, 843, 814, 708cm ^-1 ; ¹ H NMR (400MHz , DMSO-d6) δ7.88(d, J=9.0Hz, 1H), 7.61(d, J=3.1Hz, 1H), 7.41(d, J=5.0Hz, 1H), 7.17-7.05(m, 3H ), 6.97 (dd, J=5.1, 3.4Hz, 1H), 6.85 (s, 2H), 5.88 (s, 2H), 5.70 (s, 2H); ¹³ CNMR (101MHz, DMSO-d6) δ162.43, 159.09, 149.29, 141.16, 130.53, 128.79, 127.35, ^126.82 , 126.29, 121.39, 118.93, 118.03, 102.93, 102.28, 44.66; z[M+H] ⁺ Calcd for C ₁₅ H ₁₄ N ₅ S: 296.0970, Found: 296.0966.

Compound 6i, 1,3-diamino-7-ethyl-7H-pyrrolo[3,2-f]quinazoline

White solid 0.56g; yield 96%. mp 250.0-251.2°C; IR 3400, 3142, 1640, 1579, 1552, 1513, 1440, 1357, 1280, 1195, 1051, 924, 818, 704cm ^-1 ; ¹ H NMR (400MHz, DMSO-d6) δ7.78 (dd, J=9.0, 0.7Hz, 1H), 7.50(d, J=3.1Hz, 1H), 7.07(d, J=8.9Hz, 1H), 7.04(dd, J=3.1, 0.8Hz, 1H) , 6.75(s, 2H), 5.77(s, 2H), 4.27(q, J=7.2Hz, 2H), 1.38(t, J=7.2Hz, 3H); ¹³ C NMR (101MHz, DMSO-d6) δ162 .41, 159.26, 149.74, 130.36, 127.98, 121.07, 119.03, 117.65, 102.99, 101.55, 40.97, 16.36; MS (ESI) m/z: 228.1 (M ⁺ , 100); HRMS (ESI) m/z [M +H] ⁺ Calcd for C ₁₂ H ₁₄ N ₅ : 228.1249, Found: 228.1245.

The antibacterial activity test experiments of compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j will be described in detail below in conjunction with accompanying drawings 1 and 2.

In this experiment, 6 ATCC standard strains and 41 clinical strains preserved by the Chinese Academy of Medical Sciences were selected for testing. cocci and Acinetobacter baumannii. From the perspective of the epidemic trend of bacterial resistance, the tested bacteria in this test include methicillin-resistant Staphylococcus aureus (MRSA), the leading drug-resistant pathogen in nosocomial infections, and carbahydrogenase, which has the highest detection rate of clinical drug-resistant bacteria. Drug-resistant Acinetobacter baumannii (CRAB), vancomycin-resistant Enterococcus (VRE), and extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL(+)Kpn). At the same time, Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were selected as quality control bacteria.

Experimental procedure

1. Determination of minimum inhibitory concentration (MIC) by microdilution method (refer to CLSI standard)

Two days before the test, the test strains frozen in the -80°C low-temperature refrigerator were taken out, streaked on the nutrient agar plate and cultured for 18hr-24hr; the day before the test, three colonies of the same size and shape were picked and inoculated in 3mL Nutrient broth medium, constant temperature static culture at 37°C until logarithmic growth phase.

Dilute the drug stock solution twice with CAMH broth medium in a 96-well plate to form a series of required concentration gradients. Prepare to add test bacteria. Take the test bacterial solution cultivated overnight, adjust the bacterial solution to 0.5 McFarland concentration (McFarland) by turbidimetric method, and then dilute the bacterial solution adjusted to 0.5 McFarland concentration by 20 times with 0.85% normal saline, about 5× 10 ⁶ CFU/ml, draw 10 μl of the diluted bacterial solution and add it to the above series of double-diluted drug-containing CAMH broth medium. In the experiment, control tubes with no bacteria, no drug and quality control bacteria were set at the same time. After inoculation of the test bacteria, culture at a constant temperature of 37°C for 16hr-18hr. Put the 96-well plate in a light place, observe whether there is bacterial colony accumulation at the bottom of the well, and judge the end point of the test on the basis that the test results of the quality control strain fall within the quality control range. The minimum concentration of the drug contained in the sterile-grown wells is the MIC value.

2. Determination of MIC90 and MIC50 (refer to CLSI standard)

Determination of the MIC values of compounds 6d, 6e, 6f, and 6i on 41 strains of clinically isolated multidrug-resistant Acinetobacter baumannii between 2006 and 2010, the lowest drug concentration that inhibits the growth of half of the bacteria is calculated as MIC50, which will inhibit 90% The minimum drug concentration for bacterial growth is calculated as MIC90. 3. Time-kill curve determination (refer to CLSI standard)

1) The day before the test, inoculate the required test bacteria, culture and other operations are the same as those in the previous part 1 and 2.

2) The next day, take the test bacterial solution cultivated overnight, adjust the tested bacterial solution to about 10 ⁶ colony counting units (CFU/mL) by turbidimetry, and transfer it to a 50mL Erlenmeyer flask with different concentrations (1/4×MIC , 1/2×MIC, 1×MIC, 2×MIC, 4×MIC) tested antimicrobial drug mixture.

3) Take samples at 0, 2, 4, 8, and 24 hours respectively, dilute and spread on a petri dish without drugs, incubate at 35°C for 16-18 hours, calculate the average number of colonies on the plate at each concentration and time point, and take the logarithm Draw time-kill curves.

4. Determination of DHFR inhibition rate

The complete dihydrofolate reductase gene fragment was amplified by PCR, connected to the expression vector pET-30a and transformed into the expression recipient strain E.coli BL21(DE3). The inducer IPTG was added to a final concentration of 0.5 mM, 18° C., 220 rpm, and cultured with shaking for 20 h. Purification was performed using a prepacked high-efficiency Ni Sepharose affinity column.

The activity assay system is buffer solution (100 mM HEPES, 50 mM KCl, pH7.5), 5 mM 2-mercaptoethanol, 20 mM DHRF, 40 μM NADPH, 40 μM dihydrofolate. The positive control is the reaction group added with the dihydrofolate reductase inhibitor methotrexate (MTX), and the negative control is the normal reaction group without the inhibitor. The basic principle of activity measurement is that the light absorption at 340nm decreases when NADPH is reduced to NADP+, and the inhibition rate of inhibitors on DHRF is calculated according to ΔOD340. Inhibition rate=(ΔOD340nM _{without inhibitor} −ΔOD340nM _inhibitor )/ΔOD340nM _{without inhibitor} ×100%.

test results

Table 1 shows the results of the determination of the MIC value of the in vitro antibacterial activity of compounds 6a-6j against 6 ATCC standard strains, as shown in Table 1 below.

Note: ATCC43300 is methicillin-resistant Staphylococcus aureus; ATCC700603 is ESBL (+) Klebsiella pneumoniae; ATCC19606 is multidrug-resistant Acinetobacter baumannii; ATCC51299 is vancomycin-resistant Enterococcus; ATCC25922 Escherichia coli; ATCC29213 is Staphylococcus aureus

Table 1 Compound 6a-6j is to 6 ATCC standard strain antimicrobial activity MIC value determination results in vitro

Table 3 shows the in vitro antibacterial activity MIC ₅₀ and MIC ₉₀ results of the compounds and existing clinical drugs against 41 strains of clinical multidrug-resistant Acinetobacter baumannii.

Fig. 1 is the bactericidal curve of 6d to escherichia coli ATCC25922.

Fig. 2 is the bactericidal curve of 6d to Staphylococcus aureus ATCC29213.

Activity test conclusion

1. The compound has extremely low-low MIC against Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) Value (0.0004μg/mL-32μg/mL), reflecting a strong broad-spectrum antibacterial activity. Among them, the MIC values of 6a, 6b, 6c, 6d, 6e, 6f, and 6g against Staphylococcus aureus ATCC29213 were ≤0.06 μg/mL; the range of MIC values against MRSA ATCC43300 was 0.06 μg/mL-0.25 μg/mL; against Escherichia coli ATCC25922 The range of MIC value is 0.0004μg/mL-0.06μg/mL; the range of MIC value for broad-spectrum β-lactamase-producing Klebsiella pneumoniae is 0.25μg/mL-2μg/mL; The MIC of Enterococcus ATCC51299≤0.06μg/mL. The MICs of 6d and 6e against multidrug-resistant Acinetobacter baumannii ATCC19606 were 0.006μg/mL and 0.125μg/mL, respectively.

2. Compounds 6d, 6e, 6f, and 6i have MIC50 and MIC90 ranges of 8μg/mL-16μg/mL for 41 strains of clinically isolated multidrug-resistant Acinetobacter baumannii from 2006 to 2010, which are superior to most existing antibacterial drugs , and has no cross-resistance with existing antibacterial drugs such as carbatherene.

3. By testing the bactericidal curves of compound 6d on Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922, it was found that this compound has different bactericidal characteristics on G+ bacteria and G- bacteria.

4. The bactericidal curve of compound 6d on Escherichia coli ATCC25922 is shown in Figure 1. Compound 6d at different concentrations all showed antibacterial effects at 0-8 hours, and the antibacterial effects had no obvious concentration dependence, and could occur at extremely high compound concentrations. At low levels (about 2×10 ^-8 μg/mL), the maximum bactericidal effect is reached in 8-24 hours. In the concentration range of 1/8×MIC～256×MIC, the bactericidal effect can be maintained for at least 48 hours.

5. The bactericidal curve of compound 6d on Staphylococcus aureus ATCC29213 is shown in Figure 2. This compound exhibits a bactericidal effect on Staphylococcus aureus ATCC29213. The bactericidal effect can be maintained for at least 48 hours.

The above activity test results show that the compound of the new structure provided by the invention has a better inhibitory effect on various clinically infected bacteria, especially the drug-resistant bacteria in many clinical trials has a better inhibitory effect, and by contrast Experiments have proved that the compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, and 6j disclosed in the present invention have better in vitro antibacterial activity than a variety of antibacterial drugs used in the first line of clinical practice, and are more effective against a variety of drug-resistant bacteria. It has good antibacterial effect.

Claims (10)

1. The application of 1, 3-diamino-7H-pyrrole [3,2-f ] quinazoline derivatives as antibacterial drugs is disclosed, wherein the chemical structure general formula of the compounds is as follows:

wherein,

y is selected from alkyl with 1-5 carbon chains,

Z is selected from-COOCH₃、-OCH₃、-CF₃、Cl、Br、F，

R₁Selected from H, -CH₃，

R₂Selected from H, -CH₃。

2. Use of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 1 as an antibacterial agent, characterized in that:

wherein Y is selected from the group consisting of C1-3 alkanyl, p-substituted

3. Use of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 2 as an antibacterial agent, characterized in that:

wherein Z is selected from para-substituted-COOCH₃para-substituted-OCH₃para-substituted-CF₃Ortho-or para-or meta-substituted Cl, Br, F.

4. Use of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 1 as an antibacterial agent, characterized in that:

wherein R is₁、R₂Are all H.

5. Use of a 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative as an antibacterial agent according to claim 1, characterized in that it is selected from compounds of the following structure:

wherein the bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and enterococcus faecalis.

6. A medicament containing 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivatives for antibacterial use, the chemical structure of the compound being as follows:

wherein,

y is selected from alkyl with 1-5 carbon chains,

Z is selected from-COOCH₃、-OCH₃、-CF₃、Cl、Br、F，

R₁Selected from H, -CH₃，

R₂Selected from H, -CH₃。

7. A medicament containing an antibacterial use of a 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 1, characterized in that:

wherein Y is selected from the group consisting of C1-3 alkanyl, p-substituted

8. Use of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 2 as an antibacterial agent, characterized in that:

wherein Z is selected from para-substituted-COOCH₃para-substituted-OCH₃para-substituted-CF₃Ortho-or para-or meta-substituted Cl, Br, F.

9. Use of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative according to claim 1 as an antibacterial agent, characterized in that:

wherein R is₁、R₂Are all H.

10. Use of a 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative as an antibacterial agent according to claim 1, characterized in that it is selected from compounds of the following structure:

wherein the bacteria comprise Klebsiella pneumoniae ATCC700603 producing broad-spectrum β -lactamase, multidrug-resistant Acinetobacter baumannii ATCC19606, methicillin-resistant Staphylococcus aureus ATCC43300, vancomycin-resistant enterococcus faecium ATCC51299 and Escherichia coli ATCC 25922.