Design & Synthesis of Mycobacterium tuberculosis TMPK Inhibitors Yanlin Jian 21-02-2018

Evolution of Mycobacterium tuberculosis ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980

Evolution of Mycobacterium tuberculosis ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980 Robert Koch identified the specific causative agents of tuberculosis, 1882. Nobel prize, 1905.

Evolution of Mycobacterium tuberculosis Selman Waksman co-discovered streptomycin from Streptomyces griseus, the first antibiotic that could be used to cure the disease tuberculosis, 1943. Nobel prize, 1952. ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980 Robert Koch identified the specific causative agents of tuberculosis, 1882. Nobel prize, 1905.

Evolution of Mycobacterium tuberculosis Selman Waksman co-discovered streptomycin from Streptomyces griseus, the first antibiotic that could be used to cure the disease tuberculosis, 1943. Nobel prize, 1952. ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980 Robert Koch identified the specific causative agents of tuberculosis, 1882. Nobel prize, 1905. First randomized, controlled clinical trial in the practice of medicine: failure of monotherapy. Introduction of combination chemotherapy. 1946.

Evolution of Mycobacterium tuberculosis Selman Waksman co-discovered streptomycin from Streptomyces griseus, the first antibiotic that could be used to cure the disease tuberculosis, 1943. Nobel prize, 1952. MDR and XDR TB: treatment takes > 2 yrs, with expensive, toxic drugs. Since 2008 ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980 Robert Koch identified the specific causative agents of tuberculosis, 1882. Nobel prize, 1905. First randomized, controlled clinical trial in the practice of medicine: failure of monotherapy. Introduction of combination chemotherapy. 1946.

Evolution of Mycobacterium tuberculosis Selman Waksman co-discovered streptomycin from Streptomyces griseus, the first antibiotic that could be used to cure the disease tuberculosis, 1943. Nobel prize, 1952. MDR and XDR TB: treatment takes > 2 yrs, with expensive, toxic drugs. Since 2008 ~ 70000 years ago: infections of humans by M. tuberculosis 1900 1920 1940 1960 1980 5-yr follow up of patients treatment for XDR Tb in So Africa found 5% were cured, 73% died, 10% failed Rx and were discharged with positive sputa. 2014 Robert Koch identified the specific causative agents of tuberculosis, 1882. Nobel prize, 1905. First randomized, controlled clinical trial in the practice of medicine: failure of monotherapy. Introduction of combination chemotherapy. 1946.

Promising target - Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) Current Medicinal Chemistry, 2013, Vol. 20, No. 10

Promising target - Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) Key interactions: N3 in pyrimidine - Asn 109 O4 in pyrimidine - Arg 74 Fig. 1 Crystal structure of TMPKmt and TMP (PDB code 1G3U) Fig. 2 Schematic drawing of the dTMP (bulk lines) binding site of TMPKmt.

Structure - guided design of TMPKmt inhibitors A B C D S.aureus TMPK IC50 = 156 μM S.pneumoniae TMPK IC50 = 3.4 μM M. tuberculosis TMPK Ki = 1.5 μM

Structure - guided design of TMPKmt inhibitors Phenyl, pyridyl, quinolyl A B C D Different substituent aromatic ring Pyrrolyl, piperidyl Methylene, amide

Structure - guided design of TMPKmt inhibitors Phenyl, pyridyl, quinolyl A B C D Different substituent aromatic ring Pyrrolyl, piperidyl Methylene, amide Ki = 10 nM IC90 (H37Ra) = 67 μM 25

Structure - guided design of TMPKmt inhibitors Phenyl, pyridyl, quinolyl A B C D Different substituent aromatic ring Pyrrolyl, piperidyl Methylene, amide Ki = 75 nM MIC (H37Rv) = 16 - 32 μM Fig. 3 Crystal structure of TMPKmt and compound 26 26

Structure - guided design of TMPKmt inhibitors Phenyl, pyridyl, quinolyl A B C D Different substituent aromatic ring Pyrrolyl, piperidyl Methylene, amide Ki = 75 nM MIC (H37Rv) = 16 - 32 μM Fig. 4 Comparasion of crystal structures of compound 26 (yellow) and TMP (purple). 26

Siderophores - a method to increase the uptake of compounds Fig. 5 Iron uptake of bacteria by siderophore.

Siderophores - a method to increase the uptake of compounds Aiti-TB molecules Fig. 6 Trojan Horse strategy used to increase the compounds cell concentration. Chem. Rev. 2014, 114, 9154−9218

Siderophores - a method to increase the uptake of compounds Exochelin Mycobactin (amphiphilic siderophores) Carboxymycobactins (amphiphilic siderophores) D. Ferreira et al. / Journal of Proteomics 145 (2016) 153–166

Siderophores - a method to increase the uptake of compounds Artemisinin, potent antimalarial agent, No antiTB activity Mycobactin-analog-artemisinin conjugate Malarial IC50 = 0.0041 μg/ml M. tuberculosis (H37Rv) MIC = 0.39 μg/ml Mycobactin (n=14 - 21) natural siderophore J. Am. Chem. Soc. 2011, 133, 2076–2079

TMPKmt inhibitors containing siderophores 11 Entity R 1 6 2 7 3 8 4 9 5 10

TMPKmt inhibitors containing siderophores Reagents and conditions: : a) i) K2CO3, DMF, 80 ℃, 4 days, 71% yield; ii) 10% TFA/DCM, RT, 78% yield; b. SeO2, 1,4-dioxane, 99% yield; c. sodium triacetoxyborohydride, 1,2-dichloroethane, 30% yield.

TMPKmt inhibitors containing siderophores 18f 18g 18h Reagents and conditions: : a. ROH, 170-180 ℃, 0.5 h, 45% - 60% yield; b. TFA, 73 ℃, 60% - 90%.

TMPKmt inhibitors containing siderophores Reagents and conditions: a. acetic acid, 125 ℃, 2 h, 78% yield; b. TFA, 73 ℃, 70% yield.

TMPKmt inhibitors containing siderophores Reagents and conditions: a. Pd(PPh3)4, 2-(tributylstannyl)pyridine, toluene, 120 ℃, overnight, 40% yield; b. sodium triacetoxyborohydride, 1,2-dichloroethane, 79% yield; c. Methanesulfonyl chloride, Et3N, CH2Cl2, 85% yield ; d. K2CO3, MeCN, 80 ℃, 60% yield; e. TFA, 73 ℃, 61% yield.

TMPKmt inhibitors containing siderophores Entity Ki (nM) 1 640 ± 80 2 690 ± 80 3 12 ± 1 4 2500 ± 40 5 18 ± 3 6 100 ± 10 7 73 ± 6 8 15 ± 4 9 130 ± 20 10 83 ± 2 11 17600 ± 1300 3 5 8

TMPKmt inhibitors containing siderophores Entity Ki (nM) MIC (H37Rv) 1 640 ± 80 > 50 2 690 ± 80 3 12 ± 1 4 2500 ± 40 5 18 ± 3 6 100 ± 10 7 73 ± 6 8 15 ± 4 9 130 ± 20 10 83 ± 2 11 17600 ± 1300

Conclusion & Acknowledge Tuberculosis is a major cause of mortality among infectious diseases due to the emergence of MDR TB, XDR TB and increasing numbers of TB - HIV coinfections. A series of siderophore containing compounds were designed and synthesized. The siderophore containing compounds showed potent TMPKmt activity, however, none of these compounds had anti-TB activity. Acknowledge : This project is supported financially by the CSC (Chinese Scholarship Council).

Thank you

M. tuberculosis (H37Rv) MIC = 6.25 μM