Deepak Kumar a, Garima Khare b, Beena a, Saqib Kidwai c, Anil K. Tyagi b, Ramandeep Singh c and Diwan S. Rawat *a
aDepartment of Chemistry, University of Delhi, Delhi-110007, India. E-mail: dsrawat@chemistry.du.ac.in; Tel: +91-11-27667465
bDepartment of Biochemistry, University of Delhi South Campus, New Delhi-110021, India
cTranslational Health Science and Technology Institute, Vaccine and Infectious Disease Research Centre, Gurgaon, 122016, India
First published on 23rd September 2014
A series of COMPOUND LINKS
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Explore further on Open PHACTSisoniazid–amidoether derivatives was synthesized and screened for their antimycobacterial activity in vitro and in vivo. Most of the compounds exhibited potent in vitro activity against the Mycobacterium tuberculosis H37Rv strain with MIC99 values ranging from 0.39 to 3.125 μM. Five compounds were equally as potent as the reference compound COMPOUND LINKS
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Explore further on Open PHACTSisoniazid. The most active compound COMPOUND LINKS
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Download mol file of compound3b, when evaluated for in vivo activity, exhibited mild reduction in the bacillary load in lungs. However it showed a better effect in spleens. All the compounds were also evaluated for their cytotoxicity against the THP-1 cell line and no toxicity was observed up to 50 μM concentrations. The calculated ADMET parameters for the compounds validated good pharmacokinetic properties, confirming that these compounds could be used as templates for the development of new anti-tuberculosis agents.
Due to the global impact of this devastating disease, there is an urgent need for the development of new derivatives with promising antimycobacterial activities. Several different approaches such as targeting bacterial virulence, high-throughput screening (HTS), structure-based drug discovery (SBDD), chemical modifications of the known drugs and combinatorial chemistry have been explored to search novel biologically important molecules.22–24 Among all these strategies, the molecular modification approach has been found to be very promising and several drugs available in the market have been developed by using this strategy. Molecular modification is a chemical change in a molecule with the aim to enhance its pharmaceutical, pharmacokinetic or pharmacodynamic properties.
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Explore further on Open PHACTSIsoniazid (COMPOUND LINKS
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Explore further on Open PHACTSINH), a first-line anti-TB drug, is one of the most effective agents that has been used for the treatment of Mycobacterium tuberculosis infection since 1952.25,26 It is a pro-drug which is activated by the mycobacterial catalase–peroxidase enzyme known as KatG. The activated form reacts with the coenzyme NADH to form an isonicotinic acyl–COMPOUND LINKS
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Explore further on Open PHACTSNADH complex27,28 that binds with the enoyl–acyl carrier protein (ACP) reductase InhA, which is involved in the elongation of fatty acids during the mycolic acid synthesis.29 Thus COMPOUND LINKS
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Explore further on Open PHACTSisoniazid inhibits the synthesis of mycolic acid, required for the mycobacterial cell wall. COMPOUND LINKS
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Explore further on Open PHACTSINH is metabolized in the liver and forms compounds such as COMPOUND LINKS
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Explore further on Open PHACTShydrazine, which are toxic to the central nervous system and other organs.30,31 Because COMPOUND LINKS
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Explore further on Open PHACTSINH is an important drug in the therapeutic arsenal for TB treatment, efforts are being made to develop new INH derivatives with greater activity, lower toxicity and fewer side effects than COMPOUND LINKS
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Explore further on Open PHACTSINH.32–41 Several recent reports indicate that the incorporation of hydrophobic moieties into the basic structure of COMPOUND LINKS
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Explore further on Open PHACTSINH can enhance penetration of the drug into the highly lipophilic cell wall of the bacterium. Moreover, by functionalizing the hydrazine group of COMPOUND LINKS
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Explore further on Open PHACTSisoniazid and retaining its activity we can avoid the toxicity and other severe problems related to the inactivation of COMPOUND LINKS
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Explore further on Open PHACTSisoniazid by the enzyme N-acetyltransferase-2 (NAT2).
Several research groups have introduced the amido ether functionality into biologically active molecules and the resulting hybrids exhibited good biological activities.42–46 Encouraged by the previous studies and in continuation of our efforts towards the synthesis of new anti-tuberculosis agents,47–51 we proposed to attach an amido ether linkage to COMPOUND LINKS
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Explore further on Open PHACTSisoniazid to form COMPOUND LINKS
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Explore further on Open PHACTSisoniazid–amidoether derivatives and evaluated their in vitro and in vivo anti-TB activity and toxicity against THP-1 cell lines.
Scheme 1 Reagents and conditions: (i) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSEt3N, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSDCM, 0 °C to RT, 6–10 h, 70–90%; (ii) 4-hydroxybenzaldehyde, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSK2CO3, KI, RT, 10–15 h, 60–85%; (iii) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSEtOH–COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSH2O, RT, 6–10 h, 70–90%. |
Mice were tested with M. tb. H37Rv bacilli by the respiratory route in an inhalation chamber (Glascol Inc., USA) pre-calibrated to deliver approximately 1000 bacilli per animal in the lung by using frozen stocks of M. tb. H37Rv with their CFU pre-determined. Mice were euthanized on the day after infection to determine the number of CFUs implanted in the lungs. Following two weeks of infection, mice were divided into different groups – untreated, COMPOUND LINKS
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Explore further on Open PHACTSrifampicin (10 mg kg−1), and compound COMPOUND LINKS
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Download mol file of compound3b (25 mg kg−1, 50 mg kg−1, 100 mg kg−1) and therapy was initiated.
All drugs were administered once daily, five days per week, in a maximum volume of 0.175 mL by oral gavage. Before initiating the chemotherapy, the infection in the animals was verified by euthanizing a group of animals (N = 5) at 2 weeks post-infection followed by pathological observation and an enumeration of the bacillary load in the lungs and spleens. Mice (N = 5 each group) were euthanized by COMPOUND LINKS
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Explore further on Open PHACTSCO2 asphyxiation after three week, six week and ten week time points post-therapy and monitored for gross pathological observations and bacillary load. For bacterial enumeration, mice were dissected and lungs and spleens were aseptically removed and homogenized in saline. Appropriate dilutions of the homogenates were plated in duplicate onto MB7H11 agar and the plates were incubated at 37 °C for 3–4 weeks followed by an enumeration of colonies. The results were expressed as log10 CFU per organ.
At the 3 week time point, a bacillary load of 6.38 and 4.19 log10 CFU was measured in the lungs and spleens of untreated animals. The disease continued to persist and at 6 weeks post-infection also, a bacillary load of 5.91 and 3.68 log10 was recorded in the lungs and spleens of untreated animals, respectively. At 10 weeks post-infection, the disease further progressed with a bacillary load of 6.04 and 4.02 log10 in the lungs and spleens of untreated animals, respectively.
After 3 weeks of treatment with COMPOUND LINKS
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Explore further on Open PHACTSisoniazid, the CFU in the lungs of the infected animals was reduced by 1.22 log10 while after 6 weeks it showed a further CFU reduction in the bacillary load by 2.59 log10 and a further reduction of 3.38 log10 at 10 weeks when compared with the animals in the untreated group (Fig. 1A). In spleens also, COMPOUND LINKS
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Explore further on Open PHACTSisoniazid treatment resulted in a sharp reduction of bacillary load with a 2.66 log10 reduction at 3 weeks when compared with the animals in the untreated group (Fig. 1B). At 6 week and 10 week time points, no bacilli were recovered from the spleens. After 3 weeks of treatment with COMPOUND LINKS
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Explore further on Open PHACTSrifampicin, the CFU in the lungs of the infected animals was reduced by 1.93 log10 while at 6 weeks they showed a further CFU reduction in the bacillary load by 2.8 log10 when compared with the animals in the DMSO group. After 10 weeks of COMPOUND LINKS
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Explore further on Open PHACTSrifampicin administration, no bacilli were recovered from the lungs of the infected animals (Fig. 1A). In spleens also, COMPOUND LINKS
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Explore further on Open PHACTSrifampicin treatment resulted in a marked reduction in the bacillary load with a 1.76 log10 and 3.31 log10 reduction at 3 weeks and 6 weeks, respectively when compared with the animals in the DMSO group. At the 10 week time point, no bacilli were recovered from the spleens (Fig. 1B).
Fig. 1 Bacterial enumeration in lungs (A) and spleens (B) of animals belonging to different groups was carried out at various time points following infection. The bacillary load was measured as described in the Materials and methods section. Various groups are indicated. PI represents post-infection. ***, ** and * indicate statistical significance with P values <0.001, <0.01 and <0.05, respectively. The numbers in red represent the log10 CFU value by which a reduction in the particular case was observed when compared with the CFU value in the case of control animals. |
Compound COMPOUND LINKS
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Download mol file of compound3b was administered at 25 mg kg−1, 50 mg kg−1 and 100 mg kg−1 concentrations for evaluating the efficacy of this compound. When compound COMPOUND LINKS
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Download mol file of compound3b was given at a low concentration of 25 mg kg−1, it exhibited no control towards disease progression even up to 10 weeks as was evident from a comparable bacillary load observed in the case of lungs of animals treated with compound COMPOUND LINKS
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Explore further on Open PHACTSDMSO treated animals. When the concentration of compound COMPOUND LINKS
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Download mol file of compound3b was increased to 50 mg kg−1 or 100 mg kg−1 still no significant influence of the compound towards pulmonary control of the disease was observed hence, in spite of increasing the concentration up to 100 mg kg−1, no significant chemotherapeutic effect was demonstrable under our experimental conditions. At 50 mg kg−1 concentration of the compound at the 6 week time point and at 100 mg kg−1 concentration at the 10 week time point, there was a slight reduction in the pulmonary bacillary load of compound COMPOUND LINKS
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Explore further on Open PHACTSDMSO treated animals, however, the statistical significance was not of a very high order and the fact that it did not register any significant chemotherapeutic effect even at 100 mg kg−1 concentration shows that the compound had no significant intrinsic chemotherapeutic value towards the control of pulmonary tuberculosis at least in the murine model as observed in our experiments. Fig. 2 depicts the lungs of infected mice either untreated or treated with COMPOUND LINKS
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Explore further on Open PHACTSisoniazid, COMPOUND LINKS
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Explore further on Open PHACTSrifampicin and compound COMPOUND LINKS
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Fig. 2 Lungs of infected mice after treatment for 10 weeks either untreated or treated with different compounds: (A) untreated, (B) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSDMSO, (C) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSisoniazid, (D) rifampcin and (E) compound COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3b (100 mg kg−1). |
After the pulmonary infection, the settlement of bacilli in the lung tissues is followed by hematogenous spread which provides another point of control towards protection from the disease hence, the influence of compound COMPOUND LINKS
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Download mol file of compound3b was also measured on the reduction of bacillary load in spleens.
It was observed that at 25 mg kg−1 concentration of compound COMPOUND LINKS
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Download mol file of compound3b, there was a reduction in the splenic bacillary load by a value of 1.19 log10 CFU at the end of 3 weeks of chemotherapy when compared with the DMSO treated animals, however, treatment up to 10 weeks further reduced the splenic bacillary load only marginally with 1.23 log10 CFU reduction in the bacillary load at this time point (Fig. 1B). With increased concentration of compound COMPOUND LINKS
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Download mol file of compound3b to 50 mg kg−1, a more substantial effect was observed on the reduction of splenic bacillary load. We observed that at this concentration, at the end of 3 weeks of chemotherapy the splenic bacillary load exhibited a reduction by 1.4 log10 CFU. Further continuation with the chemotherapy at this dose i.e. up to 6 weeks and 10 weeks resulted in more significant reduction in the splenic bacillary load which was 2.28 log10 CFU and 2.14 log10 CFU less compared to the bacillary load in the spleens of COMPOUND LINKS
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Explore further on Open PHACTSDMSO treated animals. When the concentration of compound COMPOUND LINKS
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Download mol file of compound3b was doubled up to a concentration of 100 mg kg−1, it resulted in a more prominent bacillary load reduction in spleens and as compared to 1.4 log10 CFU reduction observed with 50 mg kg−1 concentration, this concentration resulted in a 1.77 log10 CFU reduction in the splenic bacillary load at the end of 3 weeks of treatment. Further administration of the compound to 10 weeks resulted in a very significant 3.11 log10 CFU reduction in the splenic bacillary load compared to the bacillary load in the spleens of COMPOUND LINKS
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Explore further on Open PHACTSDMSO treated animals (Fig. 1B). All the compounds were further examined for toxicity in the THP-1 cell line. The compounds were found to be non-toxic up to a concentration of 50 μM, the highest concentration tested (Table 1). It was not possible to test toxicity at higher concentrations due to solubility limitations.
Compd | Percent human oral absorptiona (>80% high, <25% poor) | QPPCacoa nm s−1 (<25 poor, >500 great) | QPlog BBa (−3.0 to 1.2) | QPPMDCKa (<25 poor, >500 great) | QPlog Khsaa (−1.5 to 1.5) | PSAa (7.0–200.0) | #Rotora (0–15) |
---|---|---|---|---|---|---|---|
a Calculated using QikProp v 3.5. Range/recommended values calculated for 95% known drugs. | |||||||
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3a | 91.362 | 373.559 | −1.66 | 170.656 | 0.195 | 107.913 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3b | 92.58 | 373.506 | −1.702 | 170.63 | 0.345 | 107.914 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3c | 94.137 | 371.023 | −1.799 | 169.404 | 0.451 | 107.915 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3d | 100 | 370.991 | −1.819 | 169.388 | 0.584 | 107.917 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3e | 93.157 | 407.696 | −1.523 | 282.274 | 0.225 | 107.448 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3f | 92.52 | 371.547 | −1.559 | 306.467 | 0.236 | 107.923 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3g | 93.934 | 373.725 | −1.557 | 308.982 | 0.235 | 107.912 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3h | 95.15 | 437.44 | −1.428 | 411.765 | 0.291 | 106.704 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3i | 94.178 | 373.705 | −1.516 | 420.73 | 0.304 | 107.912 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3j | 95.849 | 373.693 | −1.515 | 421.519 | 0.304 | 107.913 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3k | 100 | 454.177 | −1.408 | 463.239 | 0.308 | 107.1 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3l | 95.997 | 373.715 | −1.509 | 452.415 | 0.326 | 107.912 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3m | 96.867 | 373.649 | −1.509 | 453.167 | 0.327 | 107.913 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3n | 92.665 | 373.326 | −1.757 | 170.541 | 0.201 | 116.206 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3o | 72.954 | 44.669 | −2.925 | 17.186 | 0.146 | 152.833 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3p | 86.926 | 265.849 | −1.82 | 118.154 | −0.01 | 119.081 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3q | 83.384 | 202.307 | −1.976 | 87.949 | −0.116 | 120.825 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3r | 86.5 | 189.976 | −1.803 | 165.521 | 0.135 | 109.005 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3s | 87.243 | 189.824 | −1.858 | 165.336 | −0.011 | 117.291 | 10 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3t | 85.908 | 189.805 | −1.763 | 165.453 | −0.016 | 108.999 | 9 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3u | 89.562 | 210.386 | −1.86 | 165.226 | 0.088 | 109.683 | 10 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3v | 100 | 451.89 | −1.601 | 209.643 | 0.528 | 105.996 | 8 |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSIsoniazid | 66.893 | 277.461 | −0.843 | 123.742 | −0.752 | 81.355 | 2 |
All the compounds were prepared in neutralized form for the calculation of pharmacokinetic properties by Maestro Build module and LigPrep, saved in SD format. In the present study, the test compounds showed good drug-like properties based on Lipinski's rule of 5 showing zero violation of the rule, proving all the test compounds to be orally active. The descriptor QPPCaco indicating Caco-2 cells permeability, a model used for the gut–blood barrier, showed good values for all the test compounds. Similarly the values of the descriptor model such as the number of rotatable bonds (#rotor) and polar surface area (PSA), used as an indicator of bioavailability for the test compounds, lie in the expected ranges. Further, the prediction for human serum albumin binding (QPlog Khsa) and the QikProp descriptor for the brain–blood partition coefficient (QPlog BB) and the blood–brain barrier mimic MDCK cell permeability (QPPMDCK) show satisfactory predictions for all the test compounds (Table 3).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4md00288a |
This journal is © The Royal Society of Chemistry 2015 |