1. Design & Synthesis of Mycobacterium tuberculosis TMPK Inhibitors
Yanlin Jian

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

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

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

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

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

7. Evolution of Mycobacterium tuberculosis
Selman Waksman co-discovered streptomycin from Streptomyces griseus, the first antibiotic that could be used to cure the disease tuberculosis, Nobel prize, 1952.
MDR and XDR TB: treatment takes > 2 yrs, with expensive, toxic drugs. Since 2008
~ 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, Nobel prize, 1905.
First randomized, controlled clinical trial in the practice of medicine: failure of monotherapy. Introduction of combination chemotherapy

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

9. 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.

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

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

12. 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

13. 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) = μM
Fig. 3 Crystal structure of TMPKmt and compound 26 26

14. 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) = μM
Fig. 4 Comparasion of crystal structures of compound 26 (yellow) and TMP (purple). 26

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

16. 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

17. 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

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

19. TMPKmt inhibitors containing siderophores11
Entity R 1 6 2 7 3 8 4 9 5 10

20. 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.

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

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

23. 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.

24. 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

25. 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

26. 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).

27. Thank you

28. M. tuberculosis (H37Rv) MIC = 6.25 μM