CN107536835B - Application of 1, 3-diamino-7H-pyrrole [3,2-f ] quinazoline derivative as antibacterial drug and drug - Google Patents
Application of 1, 3-diamino-7H-pyrrole [3,2-f ] quinazoline derivative as antibacterial drug and drug Download PDFInfo
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Abstract
1, 3-diamino-7H-pyrrole [3,2-f]The quinazoline derivative is used as an antibacterial drug and the antibacterial drug containing the compound, and the chemical structure general formula I of the compound is as follows:
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, -CH3Wherein the bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus and enterococcus faecalis.
Description
Technical Field
The invention relates to the field of medicines, in particular to an antibacterial medical application of a 1, 3-diamino-7H-pyrrole [3,2-f ] quinazoline derivative and an antibacterial medicine containing the compound.
Background
Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate and is a precursor for the subsequent synthesis of thymidylate for purine nucleotides, methionine, serine and glycine. However, these compounds are essential starting materials for DNA, RNA and proteins required for the propagation of organisms. Thus, inhibition of the catalytic activity of dihydrofolate reductase (DHFR) can block the DNA replication process of the cell. DHFR inhibitors currently reported have a variety of biological activities, such as anti-cancer activity, anti-malarial activity, anti-mycobacterium tuberculosis activity, antibacterial activity, etc. From the chemical structure point of view, DHFR inhibitors can be divided into two types, the "classical" and the "non-classical". Classical DHFR inhibitors generally have a structure similar to folic acid, such as methotrexate; whereas the non-classical DHFR inhibitors do not have folate similarity and their core structure is the 2, 4-diaminopyrimidine structure. Compared with the classical DHFR inhibitor, the non-classical DHFR inhibitor does not have a glutamic acid residue structure in the molecule, has larger lipid solubility, can enter cells through passive diffusion, and does not need a polyglutamation process. Thus, in contrast, non-classical DHFR inhibitors can reduce the degree of resistance developed by cells, reduce toxic effects on normal cells in the human body to some extent, and can improve the effect of the drug. Such as quinazoline DHFR inhibitors pellitorine, trimetrexate and the like. They have strong DHFR inhibiting effect, high oral bioavailability, high clearance rate and little influence on histamine metabolism enzyme.
However, the classic DHFR inhibitor still has a complex structure, is difficult to synthesize, has high preparation cost and insufficient activity, particularly has a single structure, and is easy to generate new drug property.
HongWei, Wang Hao et al (Hong, W.et al, the identification of novel Mycobacterium tuberculosis DHFR inhibitors and the innovative identification of the binding of the preferences by using molecular modification. Sci.Rep.5, 15328; doi: 10.1038/srep15328, 2015) have discovered, through computer virtual screening, that 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline is a Mycobacterium tuberculosis DHFR inhibitor of the mother nucleus and a series of 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivatives were synthesized (see Wang Hao, HongWei, Oyan Kanghao, Yanghua, Yangyao, Wang Hao, Wang, Suyun, Juhua, Juju Chongli, CN106632350A, published as 2017.05.10). However, subsequent activity tests show that the antibacterial activity of the series of compounds is not very ideal, in particular the antibacterial activity against certain drug-resistant bacteria is not ideal.
Disclosure of Invention
The present invention has been made to solve the above problems, and provides a compound having a core of 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline of another structure, which has an antibacterial activity better than that of the previously disclosed analogous compounds, and in particular, has a potent multi-drug resistant acinetobacter baumannii activity, and is expected to develop a clinically useful antibacterial agent capable of inhibiting multi-drug resistant acinetobacter baumannii.
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 the content of the first and second substances,
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。
The invention provides 1, 3-diamino-7H-pyrrole [3,2-f]The use of quinazoline derivatives as antibacterial agents may also be characterised in that: wherein Y is selected from the group consisting of C1-3 alkanyl, p-substituted
The invention provides 1, 3-diamino-7H-pyrrole [3,2-f]The use of quinazoline derivatives as antibacterial agents may also be characterised in that: wherein Z is selected from para-substituted-COOCH3para-substituted-OCH3para-substituted-CF3Ortho-or para-or meta-substituted Cl, Br, F.
The invention provides 1, 3-diamino-7H-pyrrole [3,2-f]The use of quinazoline derivatives as antibacterial agents may also be characterised in that: wherein R is1、R2Are all H.
The 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivatives provided by the present invention, for use as antibacterial agents, may also be characterized by being selected from compounds of the following structures:
wherein the bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and enterococcus faecalis.
The invention also provides a medicament containing the 1, 3-diamino-7H-pyrrole [3,2-f ] quinazoline derivative for antibacterial use, wherein the chemical structure general formula I of the compound is as follows:
wherein the content of the first and second substances,
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。
The invention provides a compound containing 1, 3-diamino-7H-pyrrole [3,2-f]The antibacterial use of quinazoline derivatives as medicaments, also may have thisThe sample is characterized in that: wherein Y is selected from the group consisting of C1-3 alkanyl, p-substituted
The invention provides a compound containing 1, 3-diamino-7H-pyrrole [3,2-f]The medicaments of antibacterial use of quinazoline derivatives may also be characterised in that: wherein Z is selected from para-substituted-COOCH3para-substituted-OCH3para-substituted-CF3Ortho-or para-or meta-substituted Cl, Br, F.
The invention provides a compound containing 1, 3-diamino-7H-pyrrole [3,2-f]The medicaments of antibacterial use of quinazoline derivatives may also be characterised in that: wherein R is1、R2Are all H.
The drug for antibacterial use, which contains the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative provided by the present invention, may further have a feature characterized by being selected from compounds of the following structures:
wherein the bacteria include Klebsiella pneumoniae ATCC700603 producing broad-spectrum beta-lactamase, multi-drug resistant Acinetobacter baumannii ATCC19606, methicillin resistant Staphylococcus aureus ATCC43300, vancomycin resistant enterococcus faecium ATCC51299, and Escherichia coli ATCC 25922.
Action and Effect of the invention
The invention provides 1, 3-diamino-7H-pyrrole [3,2-f]The preparation method of quinazoline derivative adopts new raw material II and BF3The reaction is carried out, the treatment after the reaction is convenient, the product can be obtained by filtration and separation after the simple alkalization treatment, 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 the compound III is converted into the compound II, the indole is salified firstly, and then the indole reacts with dicyandiamide sodium, the invention uses organic acid p-toluenesulfonic acid as a catalyst to directly react with dicyandiamide sodium, and the method is simplified.
Further, the antibacterial activity test was performed on the obtained 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, and it was concluded that:
1. the compound has extremely low-low MIC value (0.0004 mu g/mL-32 mu g/mL) on gram-negative bacteria (escherichia coli, klebsiella pneumoniae and acinetobacter baumannii) and gram-positive bacteria (staphylococcus aureus and enterococcus faecalis), and shows strong broad-spectrum antibacterial activity. Wherein the MIC of 6a, 6b, 6c, 6d, 6e, 6f and 6g to Staphylococcus aureus ATCC29213 is less than or equal to 0.06 mu g/mL; MIC values for MRSA ATCC43300 ranged from 0.06. mu.g/mL to 0.25. mu.g/mL; the MIC value for E.coli ATCC25922 ranged from 0.0004. mu.g/mL to 0.06. mu.g/mL; the MIC value range of Klebsiella pneumoniae for producing broad-spectrum beta-lactamase is 0.25 mu g/mL-2 mu g/mL; the MIC of the vancomycin-resistant enterococcus faecium ATCC51299 is less than or equal to 0.06 mu g/mL. Wherein MICs of 6d and 6e to multidrug-resistant Acinetobacter baumannii ATCC19606 are 0.006 mu g/mL and 0.125 mu g/mL respectively. Comprehensively shows that the compounds have extremely low MIC values for gram-negative bacteria, particularly multidrug-resistant acinetobacter baumannii, and show extremely strong antibacterial activity.
2. The MIC50 and MIC90 ranges of the compound 6d, 6e, 6f and 6i to 41 strains of 2006-2010 clinically-isolated multi-drug-resistant Acinetobacter baumannii are 8-16 mu g/mL, are superior to most of existing antibacterial drugs, and have no cross resistance with existing antibacterial drugs such as hydrocarbon enzyme alkene and the like.
3. By testing the bactericidal curves of the compound 6d on staphylococcus aureus ATCC29213 and escherichia coli ATCC25922, the compound is found to have different bactericidal characteristics on G + bacteria and G-bacteria.
4. The sterilization curve of the compound 6d on escherichia coli ATCC25922 shows that the compound 6d with different concentrations shows a bacteriostatic effect within 0-8 hr, and the bacteriostatic effect has no obvious concentration dependence and can occur under the condition that the concentration of the compound is extremely low(about 2X 10)-8Mu g/mL) in 8-24 hr to obtain the maximum sterilization effect. The bactericidal effect can be maintained for at least 48hr within the concentration range of 1/8 × MIC-256 × MIC.
5. The sterilization curve of the compound 6d on staphylococcus aureus ATCC29213 shows that the compound has a sterilization effect on staphylococcus aureus ATCC29213, the sterilization is carried out quickly within 4-8 hours, and the sterilization effect can be maintained at least for 48 hours within the concentration of 1/4 XMIC to 4 XMIC.
The activity tests show that the compound provided by the invention has better inhibition effect on various bacteria causing infection clinically, especially on multi-drug resistant bacteria in clinic, and the control experiments prove that the in vitro antibacterial activity of the disclosed compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i and 6j is superior to that of various antibacterial drugs used in the first line of clinic and has better antibacterial effect on various drug resistant bacteria.
Drawings
FIG. 1 is a graph of the bactericidal profile of 6d against E.coli ATCC 25922; and
FIG. 2 is a graph of the bactericidal profile of 6d against Staphylococcus aureus ATCC 29213.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the following examples describe the preparation method of the 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative and the physicochemical and spectral data of the compound in detail.
Compound route:
experimental part:
general procedure for the Synthesis of Compounds 3 a-j: 5-Nitroindole (6.17mmol) was added to anhydrous DMSO (30ml), 60% NaH (7.40mmol) was added in portions under argon, and the mixture was stirred at room temperature for 1 h. Then, the compounds 2a to j (7.40mmol) were added to the mixture, and the reaction was stirred at room temperature for 3 hours. The reaction was quenched by addition of 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 removed by evaporation under reduced pressure to give a crude product.
General procedure for the Synthesis of Compounds 4 a-j: compounds 3a-j (4.28mmol), iron powder (21.41mmol) and ammonium chloride (42.82mmol) were added to ethanol-water (4: 1) (50ml) and the reaction was stirred at 80 ℃ under reflux for 4 h. And (3) carrying out rotary evaporation on the reaction solution, adding distilled water into the residue for dissolving, adjusting the pH value to be alkaline by using anhydrous sodium bicarbonate, extracting by using dichloromethane, washing an organic layer by using distilled water, drying by using anhydrous sodium sulfate, and removing the solvent by reduced pressure evaporation to obtain a crude product.
General procedure for the Synthesis of Compounds 5 a-j: compound 4a-j (3.12mmol) was dissolved in anhydrous DMF (10ml) and p-toluenesulfonic acid (3.12mmol) and NaN (CN) were added2(9.21mmol), the reaction was stirred at 50 ℃ overnight. Pouring the reaction solution into water of which the amount is about 4 times that of the solvent, stirring, performing suction filtration, and drying a filter cake to obtain a crude product.
General procedure for the Synthesis of Compounds 6 a-j: compounds 5a-j (2.58mmol) were added to DME (15ml) and BF added under ice bath conditions3-Et2O (12.91mmol), the reaction was stirred at 60 ℃ overnight. And (3) carrying out rotary evaporation on the reaction solution, dissolving residues in a small amount of methanol, adding 1mol/L NaOH solution, stirring, carrying out suction filtration, and drying a filter cake to obtain a crude product. Purification by column chromatography eluting with dichloromethane: methanol 20: 1 (V: V) gave the product.
The compound 6a, 1, 3-diamino-7- (4-methoxycarbonyl-benzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.82g of white solid; the yield thereof was found to be 92%. mp 173.6-174.8 ℃; IR 3503, 3458, 3316, 3148, 1703, 1632, 1577, 1556, 1518, 1476, 1358, 1289, 935, 818, 741 cm-1；1HNMR(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＝3.1 Hz，1H)，7.03(d，J＝9.0Hz，1H)，6.83(s，2H)，5.83(s，2H)，5.62(s，2H)， 3.82(s，3H)；13CNMR(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；MS(ESI)m/z：348.1(M+，100)； HRMS(ESI)m/z[M+H]+Calcd for C19H18N5O2：348.1460，Found：348.1455。
The compound 6b, 1, 3-diamino-7- (4-trifluoromethyl-benzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.85g of white solid; the yield thereof was found to be 92%. mp 143.8-144.8 ℃; IR 3326, 3171, 1583, 1519, 1495, 1358, 1326, 1111, 1066, 1017, 936, 819, 750, 716cm "1; 1H NMR (400MHz, DMSO-d6) δ 7.76(d, J ═ 9.0Hz, 1H), 7.72-7.66(m, 3H), 7.34(d, J ═ 8.0 Hz, 2H), 7.19(d, J ═ 3.1Hz, 1H), 7.06(d, J ═ 9.0Hz, 1H), 6.99(s, 2H), 6.01(s, 2H), 5.65(s, 2H); 13C NMR (101 MHz, DMSO-d6) δ 162.51, 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 ] + Calcdfor C18H15F3N 5: 358.1280, Found: 358.1287.
the compound 6c, 1, 3-diamino-7- (4-bromobenzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.85g of white solid; the yield thereof was found to be 90%. mp 224.1-225.4 ℃; IR 3425, 3074, 1588, 1519, 1490, 1466, 1439, 1360, 1294, 1274, 1071, 1040, 1011, 938, 817, 798, 758, 718cm-1；1H 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)；13C 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 C17H15BrN5：368.0511，Found：368.0510。
The compound 6d, 1, 3-diamino-7- (4-fluorobenzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.75g of white solid; the yield thereof was found to be 95%. mp179.8-181.2 ℃; IR 3320, 3167, 1620, 1581, 1555, 1510, 1442, 1357, 1272, 1222，1157，1067，936，820，725 cm-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＝8.9 Hz，1H)，6.93(s，2H)，5.96(s，2H)，5.50(s，2H)；13C 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]+Calcd for C17H15FN5：308.1311，Found：308.1309。
The compound 6e, 1, 3-diamino-7- (3-fluorobenzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.73g of white solid; the yield thereof was found to be 92%. mp 229.9-231.6 ℃; IR 3425, 3076, 1620, 1588, 1519, 1494, 1463, 1435, 1357, 1279, 1246, 1189, 1132, 1074, 937, 818, 780, 715cm-1；1H 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(101 MHz，DMSO-d6)δ163.88，162.48，158.92，148.81， 141.62，131.08，130.78，129.52，123.39，123.37，121.39，118.11，114.79， 114.02，102.87，102.21，49.14；MS(ESI)m/z：308.1(M+，100)；HRMS (ESI)m/z[M+H]+Calcd for C17H15FN5：308.1311，Found：308.1311。
The compound 6f, 1, 3-diamino-7- (2-fluorobenzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.70g of white solid; the yield thereof was found to be 88%. mp 193.0-194.6 ℃; IR 3428, 3082, 1620, 1584, 1519, 1489, 1357, 1272, 1037, 934, 835, 817, 754, 703cm-1；1H NMR(400MHz，DMSO-d6)δ7.81(d，J＝9.0Hz，1H)，7.62(d，J＝3.1 Hz，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)；13C NMR(101 MHz，DMSO-d6) δ162.54，158.60，148.09，130.97，130.26，129.74，129.64，125.44，125.30， 125.15，121.28，118.18，116.01，115.80，102.80，102.23，46.17；MS(ESI) m/z：308.1(M+，100)；HRMS(ESI)m/z[M+H]+Calcd for C17H15FN5： 308.1311，Found：308.1317。
The compound 6g, 1, 3-diamino-7- (4-methoxy-benzyl) -7H-pyrrolo [3,2-f ] quinazoline
0.76g of white solid; the yield thereof was found to be 92%. mp 184.3-185.5 ℃; IR 3490, 3338, 3197, 1661, 1583, 1549, 1516, 1494, 1358, 1253, 1177, 1031, 936, 816, 763, 702cm-1；1H 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＝ 8.9Hz，1H)，6.92-6.84(m，2H)，6.32(s，2H)，5.44(s，2H)，3.70(s，3H)；13C NMR(101 MHz，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)m/z [M+H]+Calcd for C18H18N5O：320.1511，Found：320.1513。
The compound 6H, 1, 3-diamino-7- (cyclopropylmethyl) -7H-pyrrolo [3,2-f ] quinazoline
0.59g of white solid; the yield thereof was found to be 90%. mp 200.0-201.8 deg.C; IR 3479, 3384, 3124, 1653, 1625, 1578, 1554, 1519, 1494, 1459, 1361, 1264, 1244, 1118, 928, 818, 722cm-1；1H 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)；13C 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 C14H16N5：254.1406，Found：254.1402。
The compound 6i, 1, 3-diamino-7- (thiophene-2-methyl) -7H-pyrrolo [3,2-f ] quinazoline
0.70g of white solid; the yield thereof was found to be 92%. mp 184.5-185.6 ℃; IR 3435, 3311, 3127, 1617, 1584, 1515, 1489, 1453, 1431, 1356, 1239, 1187, 1120, 1036, 933, 843, 814, 708cm-1；1H 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)；13C NMR(101 MHz，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；MS(ESI)m/z：296.1(M+，100)；HRMS(ESI) m/z[M+H]+Calcd for C15H14N5S：296.0970，Found：296.0966。
The compound 6i, 1, 3-diamino-7-ethyl-7H-pyrrolo [3,2-f ] quinazoline
0.56g of white solid; the yield thereof was found to be 96%. mp 250.0-251.2 deg.C; IR 3400, 3142, 1640, 1579, 1552, 1513, 1440, 1357, 1280, 1195, 1051, 924, 818, 704cm-1；1H 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)；13C NMR(101 MHz，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 C12H14N5： 228.1249，Found：228.1245。
The tests for testing the antibacterial activity of the compounds 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i and 6j will be described in detail with reference to FIGS. 1 and 2.
In the test, 6 ATCC standard strains and 41 clinical strains stored in the Chinese medical academy of sciences are selected for testing, and the tested strains basically comprise clinical common pathogenic bacteria: escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, enterococcus faecalis, and Acinetobacter baumannii. From the epidemic trend of bacterial drug resistance, the tested bacteria of the test comprise methicillin-resistant staphylococcus aureus (MRSA) infected with the primary drug-resistant pathogenic bacteria in hospital, carbohydrogenarene-resistant acinetobacter baumannii (CRAB) with the highest detection rate of clinical drug-resistant bacteria, vancomycin-resistant enterococcus (VRE) and klebsiella pneumoniae (ESBL (+) Kpn) producing broad-spectrum beta-lactamase. Meanwhile, Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 are selected as quality control bacteria.
Procedure of the test
1. Measurement of Minimum Inhibitory Concentration (MIC) by microdilution method (reference CLSI Standard)
Taking out the strain frozen in refrigerator at-80 deg.C two days before test, and streaking on nutrient agar culture dish for resuscitation and culture for 18hr-24 hr; three colonies with consistent size and morphology were picked up and inoculated in 3mL nutrient broth medium on the day before the experiment, and the colonies were cultured at constant temperature of 37 ℃ until the logarithmic phase.
Drug stock was diluted twice in 96-well plates with CAMH broth to a series of desired concentration gradients, final concentrations 1024 μ g/mL, 512 μ g/mL, 256 μ g/mL. Taking overnight-cultured test bacterial liquid, adjusting bacterial liquid to 0.5 McFarland concentration by using turbidimetric method, and diluting bacterial liquid adjusted to 0.5 McFarland concentration by 20 times with 0.85% physiological saline, wherein the dilution is about 5 × 106CFU/ml, sucking 10 μ l of diluted bacterial liquid, adding into the above series of two-fold diluted medicinal CAMH broth culture medium, and setting no-bacteria, no-medicine and quality control bacteria control tubes during the experiment. Inoculating test bacteria, and culturing at 37 deg.C for 16hr-18 hr. And (3) placing the 96-well plate in a light place, observing whether bacterial colony deposition is accumulated at the bottom of the hole, and judging a test end point on the basis that a test result of the quality control bacterial strain falls within a quality control range. The minimum concentration of drug contained in the aseptically grown wells was the MIC value.
MIC90, MIC50 assay (reference CLSI Standard)
The MIC values of compound 6d, 6e, 6f, 6i, for the 41 clinically isolated multidrug-resistant a. baumannii strains between 2006-2010 were determined, with the lowest drug concentration that inhibited half of the bacterial growth being MIC50 and the lowest drug concentration that inhibited 90% of the bacterial growth being MIC 90.
3. Sterilization Curve (Time-kill curve) determination (refer to CLSI Standard)
1) The operation of inoculating the required test bacteria on the day before the test, culturing and the like is the same as the operation of the previous parts 1 and 2.
2) The next day, the test bacterial liquid cultured overnight was taken and the test bacterial liquid was adjusted to about 10 degrees centigrade by turbidimetry6Colony counting units (CFU/mL) were transferred to 50mL flasks and mixed with varying concentrations (1/4 × MIC, 1/2 × MIC, 1 × MIC, 2 × MIC, 4 × MIC) of the test antibacterial agent.
3) Sampling at 0, 2, 4, 8, and 24hr respectively, diluting and spreading in culture dish without medicine, culturing at 35 deg.C for 16-18hr, calculating average colony number of the dish at each concentration and time point, taking logarithm, and plotting time-sterilization curve.
Measurement of DHFR inhibition Rate
The complete dihydrofolate reductase gene fragment is amplified by PCR, connected with an expression vector pET-30a and transformed to express a receptor bacterium E.coli BL21(DE 3). The inducer IPTG was added to a final concentration of 0.5mM, 18 ℃ and 220rpm, and shake-cultured for 20 hours. Purifying by high-efficiency Ni Sepharose affinity column.
The activity assay was buffer (100mM HEPES, 50mM KCl, pH7.5), 5mM 2-mercaptoethanol, 20mM DHRF, 40. mu.M NADPH, 40. mu.M dihydrofolate. The positive control is a reaction group to which Methotrexate (MTX), a dihydrofolate reductase inhibitor, is added, and the negative control is a normal reaction group to which no inhibitor is added. The basic principle of the activity assay is that the absorbance at 340nm is reduced when NADPH is reduced to NADP +, and the inhibition rate of the inhibitor on DHRF is calculated from the Δ OD 340. Inhibition rate (Δ OD340 nM)Non-inhibitor-ΔOD340nMInhibitors) /ΔOD340nMNon-inhibitor×100％。
Test results
Table 1 shows the results of MIC values of 6a-6j for 6 ATCC standard strains in vitro, as shown in Table 1 below.
Note: ATCC43300 is methicillin resistant staphylococcus aureus; ATCC700603 is Klebsiella pneumoniae from ESBL (+); ATCC19606 is multidrug resistant acinetobacter baumannii; ATCC51299 is vancomycin-resistant enterococcus; ATCC25922 is escherichia coli; ATCC29213 Staphylococcus aureus
TABLE 1 MIC value determination of in vitro antibacterial Activity of Compounds 6a-6j against 6 ATCC Standard strains
Determine the result
Table 3 shows MIC of in vitro antibacterial activity of compounds and existing clinical drugs on 41 clinical multi-drug resistant Acinetobacter baumannii strains50、MIC90And (4) value measurement results.
FIG. 1 is a graph of the bactericidal profile of 6d against E.coli ATCC 25922.
FIG. 2 is a graph of the bactericidal profile of 6d against Staphylococcus aureus ATCC 29213.
Conclusion of Activity test
1. The compound has extremely low-low MIC value (0.0004 mu g/mL-32 mu g/mL) on gram-negative bacteria (escherichia coli, klebsiella pneumoniae and acinetobacter baumannii) and gram-positive bacteria (staphylococcus aureus and enterococcus faecalis), and shows strong broad-spectrum antibacterial activity. Wherein the MIC of 6a, 6b, 6c, 6d, 6e, 6f and 6g to Staphylococcus aureus ATCC29213 is less than or equal to 0.06 mu g/mL; MIC values for MRSA ATCC43300 ranged from 0.06. mu.g/mL to 0.25. mu.g/mL; the MIC value for E.coli ATCC25922 ranged from 0.0004. mu.g/mL to 0.06. mu.g/mL; the MIC value range of Klebsiella pneumoniae for producing broad-spectrum beta-lactamase is 0.25 mu g/mL-2 mu g/mL; the MIC of the vancomycin-resistant enterococcus faecium ATCC51299 is less than or equal to 0.06 mu g/mL. Wherein MICs of 6d and 6e to multidrug-resistant Acinetobacter baumannii ATCC19606 are 0.006 mu g/mL and 0.125 mu g/mL respectively.
2. The compound 6d, 6e, 6f, 6i has the MIC50 and MIC90 ranges from 8 mu g/mL to 16 mu g/mL for 41 strains of 2006-2010 clinically-isolated multi-drug-resistant acinetobacter baumannii, is superior to most of the existing antibacterial drugs, and has no cross resistance with the existing antibacterial drugs such as hydrocarbon enzyme alkene and the like.
3. By testing the bactericidal curves of the compound 6d on staphylococcus aureus ATCC29213 and escherichia coli ATCC25922, the compound is found to have different bactericidal characteristics on G + bacteria and G-bacteria.
4. The bactericidal curve of the compound 6d on escherichia coli ATCC25922 is shown in figure 1, the compound 6d with different concentrations shows bacteriostatic effect within 0-8 hr, and the bacteriostatic effect has no obvious concentration dependence and can occur under the condition of extremely low compound concentration (about 2 multiplied by 10)-8Mu g/mL) in 8-24 hr to obtain the maximum sterilization effect. The bactericidal effect can be maintained for at least 48hr within the concentration range of 1/8 × MIC-256 × MIC.
5. The sterilization curve of the compound 6d on staphylococcus aureus ATCC29213 is shown in figure 2, the compound has a sterilization effect on staphylococcus aureus ATCC29213, the sterilization is carried out quickly within 4-8 hours, and the sterilization effect can be maintained at least for 48 hours within the concentration range of 1/4 XMIC to 4 XMIC.
The activity test results show that the compound with the novel structure has better inhibition effect on various bacteria causing infection clinically, particularly drug-resistant bacteria in multiple clinics, and the in vitro antibacterial activity of the disclosed compound 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i and 6j is proved to be superior to various antibacterial drugs used in the first line of clinic and has better antibacterial effect on various drug-resistant bacteria through contrast experiments.
Claims (1)
- Use of a 1, 3-diamino-7H-pyrrolo [3,2-f ] quinazoline derivative in the preparation of an antibacterial medicament, characterised in that the compound is selected from the following structures:
among them, the bacterium is Escherichia coli ATCC 25922.
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| US4118561A (en) * | 1977-04-06 | 1978-10-03 | American Home Products Corporation | 7-(Substituted)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamines |
| US4208520A (en) * | 1978-06-28 | 1980-06-17 | American Home Products Corporation | 7-(Substituted)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamines |
| CN106632350A (en) * | 2016-11-16 | 2017-05-10 | 宁夏医科大学 | 1,3-diamido-7H-pyrrole[3,2-f] quinazoline derivative and preparation method thereof |
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| US4118561A (en) * | 1977-04-06 | 1978-10-03 | American Home Products Corporation | 7-(Substituted)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamines |
| US4208520A (en) * | 1978-06-28 | 1980-06-17 | American Home Products Corporation | 7-(Substituted)-7H-pyrrolo[3,2-f]quinazoline-1,3-diamines |
| CN106632350A (en) * | 2016-11-16 | 2017-05-10 | 宁夏医科大学 | 1,3-diamido-7H-pyrrole[3,2-f] quinazoline derivative and preparation method thereof |
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