Nishad
Thamban Chandrika‡
,
Keith D.
Green‡
,
Abbygail C.
Spencer
,
Oleg V.
Tsodikov
and
Sylvie
Garneau-Tsodikova
*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596, USA. E-mail: sylviegtsodikova@uky.edu
First published on 14th June 2023
Novel substituted monohydrazides synthesized for this study displayed broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains. The activity of these compounds was either comparable or superior to amphotericin B against most of the fungal strains tested. These compounds possessed fungistatic activity in a time-kill assay and exhibited no mammalian cell toxicity. In addition, they prevented the formation of fungal biofilms. Even after repeated exposures, the Candida albicans ATCC 10231 (strain A) fungal strain did not develop resistance to these monohydrazides.
Despite the increasing threat of spread of antimicrobial-resistant fungi, treatment options remain limited.22,23 The three available classes of current antifungals: the azoles, such as fluconazole (FLC) and voriconazole (VRC), the echinocandins, such as caspofungin (CFG), and the polyenes, such as amphotericin B (AmB) are still staple treatments of IFIs. Overreliance on these agents and their excessive use have caused an increase in the percentage of fungal species resistant to these classes of drugs.24,25 IFIs have also emerged as the leading co-infection in patients hospitalized with severe COVID-19 infections.26–29 The cost of antimicrobial resistance is immense, both for the economy and the human health.30–32 The narrow spectrum of antifungal activity, limited efficacy, significant side effects, drug–drug interactions, and toxicity associated with existing antifungal drugs highlight the urgent need to develop novel antifungal therapeutic agents.33–35
In recent years, our group has been actively involved in developing novel small molecules as well aminoglycoside-based antifungal agents to treat both topical and systemic fungal infections.36–50 We previously reported the development of bis(N-amidinohydrazones), N-(amidino)-N′-aryl-bishydrazones, and N,N′-diaryl bishydrazones as potential antibacterial and antifungal agents.51,52 Next, we explored the efficacy of monohydrazones over bishydrazones.53 With the promising results we observed for monohydrazones, we now decided to explore novel substituted monohydrazides as potential antifungals. Herein, we report the synthesis and antifungal activity of 64 novel substituted monohydrazides by in vitro studies as well as by time-kill studies. We also explore their efficacy against biofilms. Finally, we investigate their toxicity profile against mammalian cell lines and their potential susceptibility to the development of resistance by C. albicans.
Fig. 1 The synthetic scheme for the preparation of the compounds used in this study. Note: the chemical yields are provided in parentheses. |
Fungal strain | |||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Candida albicans | Non-albicans Candida | Non-Candida | |||||||||||||||||
Cpd # | A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S |
Strains: A = C. albicans ATCC 10231, B = C. albicans ATCC 64124, C = C. albicans ATCC MYA-2876(S), D = C. albicans ATCC 90819(R), E = C. albicans ATCC MYA-2310(S), F = C. albicans ATCC MYA-1237(R), G = C. albicans ATCC MYA-1003(R), H = C. glabrata ATCC 2001, I = C. krusei ATCC 6258, J = C. parapsilosis ATCC 22019, K = C. auris AR Bank # 0384, L = C. auris AR Bank # 0390, M = C. neoformans ATCC MYA-895, N = A. terreus ATCC MYA-3633, O = A. flavus ATCC MYA-3631, P = A. nidulans ATCC 38163, Q = A. fumigatus NRRL 163, R = A. fumigatus NRRL 5109, and S = A. fumigatus NRRL 6113. Note: here, the (S) and (R) indicate that ATCC reports these strains to be susceptible (S) and resistant (R) to itraconazole (ITC) and fluconazole (FLC). | |||||||||||||||||||
1a | 0.24 | 1.95 | 3.9 | 7.8 | 3.9 | 0.98 | 1.95 | 7.8 | 1.95 | 3.9 | 1.95 | 0.24 | 1.95 | 1.95 | >31.3 | 3.9 | >31.3 | >31.3 | >31.3 |
1d | 0.49 | 7.8 | 7.8 | 1.95 | 3.9 | 1.95 | 1.95 | 7.8 | 1.95 | 15.6 | 1.95 | 1.95 | 0.49 | 3.9 | >31.3 | 3.9 | >31.3 | >31.3 | >31.3 |
1f | 0.98 | 1.95 | 1.95 | 1.95 | 1.95 | 1.95 | 1.95 | 3.9 | 0.98 | 7.8 | 1.95 | 1.95 | 0.98 | 31.3 | >31.3 | 1.95 | >31.3 | >31.3 | >31.3 |
2a | ≤0.06 | 0.98 | 0.98 | 0.49 | 1.95 | 0.98 | 0.98 | 1.95 | 0.24 | 1.95 | 1.95 | 0.24 | 0.12 | 1.95 | 7.8 | 0.49 | >31.3 | >31.3 | >31.3 |
2b | 0.12 | 0.98 | 0.98 | 0.49 | 0.98 | 0.98 | 0.49 | 1.95 | 0.49 | 1.95 | 0.98 | 0.98 | 0.12 | 0.98 | >31.3 | 0.24 | >31.3 | >31.3 | >31.3 |
2c | 0.24 | 0.98 | 0.98 | 1.95 | 1.95 | 1.95 | 0.98 | 15.6 | 0.49 | 3.9 | 0.98 | 0.98 | 0.24 | 3.9 | >31.3 | 1.95 | 7.8 | 3.9 | >31.3 |
2d | 0.12 | 0.98 | 3.9 | 0.98 | 1.95 | 1.95 | 0.49 | 1.95 | 0.12 | 1.95 | 3.9 | 0.98 | 0.24 | 1.95 | 31.3 | 0.49 | 31.3 | 7.8 | 15.6 |
2e | 0.12 | 0.98 | 1.95 | 0.24 | 0.98 | 0.98 | 0.98 | 1.95 | 1.95 | 3.9 | 1.95 | 1.95 | 0.49 | 3.9 | 31.3 | 0.49 | >31.3 | 7.8 | 15.6 |
2f | 0.12 | 0.98 | 1.95 | 0.49 | 0.98 | 0.98 | 0.98 | 0.98 | 0.49 | 3.9 | 3.9 | 0.12 | 0.49 | 31.3 | >31.3 | 0.49 | >31.3 | >31.3 | 31.3 |
2g | 0.12 | 1.95 | 0.98 | 0.98 | 31.3 | 0.49 | 0.49 | 0.98 | 0.49 | 1.95 | 0.49 | 0.12 | 0.24 | 0.98 | 15.6 | 0.49 | >31.3 | 15.6 | 31.3 |
2h | 0.12 | 1.95 | 3.9 | 0.98 | 1.95 | 1.95 | 0.98 | 3.9 | 0.24 | 3.9 | 0.24 | 0.98 | 0.49 | 0.98 | 3.9 | 0.49 | >31.3 | 15.6 | >31.3 |
2i | ≤0.06 | 0.98 | 0.98 | 0.98 | 0.98 | 1.95 | 0.49 | 7.8 | 0.24 | 1.95 | 0.24 | 0.49 | 0.24 | 0.98 | 1.95 | 0.49 | >31.3 | >31.3 | 31.3 |
2j | 0.24 | 1.95 | 1.95 | 0.98 | 1.95 | 3.9 | 1.95 | 7.8 | 0.98 | 1.95 | 7.8 | 1.95 | 0.49 | 1.95 | >31.3 | 0.49 | >31.3 | >31.3 | >31.3 |
3a | 0.49 | 0.49 | 3.9 | 3.9 | 3.9 | 3.9 | 1.95 | 31.3 | 0.24 | 1.95 | 1.95 | 1.95 | ≤0.06 | 1.95 | 7.8 | 0.98 | >31.3 | >31.3 | >31.3 |
3c | 0.24 | 0.98 | 0.49 | 3.9 | 1.95 | 1.95 | 0.98 | 15.6 | 0.24 | 3.9 | 1.95 | 0.98 | 0.24 | 3.9 | >31.3 | 1.95 | >31.3 | >31.3 | >31.3 |
3f | 0.24 | 0.98 | 0.98 | 0.98 | 0.98 | 0.49 | 0.49 | 1.95 | 0.24 | 3.9 | 3.9 | 0.49 | ≤0.06 | 31.3 | >31.3 | 1.95 | >31.3 | >31.3 | >31.3 |
4a | 0.49 | 0.49 | 1.95 | 3.9 | 3.9 | 3.9 | 0.98 | >31.3 | 0.24 | 7.8 | 1.95 | 1.95 | 0.06 | 0.98 | 15.6 | 1.95 | >31.3 | >31.3 | >31.3 |
4c | 0.98 | 3.9 | 0.98 | 1.95 | 3.9 | 0.98 | 3.9 | 3.9 | 0.24 | 1.95 | 0.98 | 1.95 | 0.49 | 1.95 | >31.3 | 3.9 | >31.3 | 15.6 | >31.3 |
5c | 0.98 | 0.98 | 0.49 | 3.9 | 1.95 | 1.95 | 0.98 | 15.6 | 0.24 | 3.9 | 1.95 | 0.98 | 0.49 | 3.9 | >31.3 | 1.95 | 15.6 | 7.8 | >31.3 |
5d | 0.24 | 0.98 | 1.95 | 1.95 | 3.9 | 1.95 | 0.98 | 15.6 | 0.49 | 3.9 | 1.95 | 0.98 | 0.49 | 1.95 | 7.8 | 0.98 | 31.3 | 15.6 | 15.6 |
5f | 0.24 | 1.95 | 0.98 | 1.95 | 0.98 | 0.98 | 0.98 | 0.98 | 0.49 | 7.8 | 1.95 | 1.95 | 0.24 | 15.6 | >31.3 | 1.95 | >31.3 | >31.3 | 15.6 |
6c | 1.95 | 3.9 | 0.49 | 1.95 | 7.8 | 0.98 | 3.9 | 7.8 | 0.24 | 0.98 | 0.98 | 1.95 | 0.98 | 1.95 | >31.3 | 3.9 | >31.3 | 31.3 | >31.3 |
7a | 0.24 | 0.98 | 1.95 | 3.9 | 1.95 | 3.9 | 1.95 | 31.3 | 0.24 | 1.95 | 1.95 | 1.95 | ≤0.06 | 3.9 | >31.3 | 0.98 | >31.3 | >31.3 | >31.3 |
7d | 0.98 | 1.95 | 1.95 | 1.95 | 3.9 | 1.95 | 0.98 | 15.6 | 0.49 | 3.9 | 1.95 | 0.98 | 0.49 | 3.9 | 7.8 | 0.49 | >31.3 | >31.3 | >31.3 |
7f | 1.95 | 1.95 | 1.95 | 1.95 | 1.95 | 0.98 | 0.98 | 7.8 | 0.98 | 7.8 | 3.9 | 0.98 | 1.95 | 31.3 | >31.3 | 1.95 | 31.3 | 31.3 | 31.3 |
8a | 0.98 | 0.98 | 1.95 | 3.9 | 1.95 | 1.95 | 0.98 | 15.6 | 0.49 | 7.8 | 1.95 | 1.95 | ≤0.06 | 3.9 | >31.3 | 1.95 | >31.3 | >31.3 | >31.3 |
8d | 0.24 | 0.98 | 0.98 | 0.98 | 0.98 | 0.98 | 0.49 | 15.6 | 0.24 | 7.8 | 1.95 | 0.98 | 0.49 | 1.95 | 7.8 | 0.49 | 31.3 | 15.6 | >31.3 |
8f | 1.95 | 1.95 | 1.95 | 1.95 | 1.95 | 0.98 | 0.98 | 7.8 | 0.98 | 7.8 | 3.9 | 0.98 | 0.98 | 31.3 | >31.3 | 1.95 | 31.3 | 15.6 | 31.3 |
9a | 0.24 | 1.95 | 3.9 | 0.98 | 7.8 | 0.98 | 0.98 | 7.8 | 0.49 | 3.9 | 0.98 | 0.98 | 0.12 | 0.98 | 15.6 | 0.98 | >31.3 | >31.3 | >31.3 |
9b | 0.24 | 1.95 | 1.95 | 3.9 | 7.8 | 0.98 | 0.98 | 3.9 | 0.24 | 1.95 | 1.95 | 0.98 | 0.24 | 0.98 | >31.3 | 0.98 | >31.3 | >31.3 | >31.3 |
9c | 0.49 | 0.98 | 1.95 | 1.95 | 1.95 | 1.95 | 0.98 | 7.8 | 0.49 | 1.95 | 1.95 | 0.98 | 0.49 | 1.95 | >31.3 | 1.95 | 15.6 | 7.8 | >31.3 |
9f | 0.49 | 0.98 | 1.95 | 0.98 | 1.95 | 0.98 | 1.95 | 0.98 | 0.98 | 3.9 | 0.49 | 0.49 | 0.98 | >31.3 | >31.3 | 0.98 | 31.3 | 31.3 | 31.3 |
9g | 0.24 | 0.98 | 0.98 | 0.98 | 3.9 | 0.49 | 0.49 | 0.98 | 0.24 | 3.9 | 3.9 | 0.98 | 0.24 | 0.98 | 7.8 | 0.49 | >31.3 | >31.3 | >31.3 |
AmB | 1.95 | 1.95 | 1.95 | 0.98 | 0.98 | 1.95 | 0.98 | 0.98 | 0.98 | 0.98 | 1.95 | 1.95 | 7.8 | 3.9 | 31.3 | 15.6 | 0.98 | 0.98 | 1.95 |
From the data reported in Table 1 and S1,† we observed that compounds from series 2 and series 9 performed better compared to those in the other seven series of compounds against the sixteen strains (A–P) tested. A detailed analysis of the nine series (i.e., series 1–9) led to the following conclusions. In the case of monohydrazides 1a–1j (i.e., R1 = 2,4-diF, X = C, Y = C; Fig. 1), compounds 1h, 1i, and 1j generally displayed poor activity against strains A–P, with the exception of the following combinations; 1h (MIC = 0.98 μg mL−1 against strain M), 1i (MIC = 1.95 μg mL−1 and 0.49 μg mL−1 against strains I and M, respectively), and 1j (MIC = 1.95 μg mL−1 against strain I). Among series 1, compounds 1f, 1a, 1d, and 1c performed better with excellent activity (0.24–1.95 μg mL−1) against twelve strains (A–G, I, K–M, and P), nine strains (A, B, F, G, I, and K–N), eight strains (A, D, F, G, I, and K–M), and seven strains (A, B, G, I, and K–M), respectively. Monohydrazides 2a–2j (i.e., R1 = H, X = N, Y = C; Fig. 1) displayed excellent to good activity (0.06–7.8 μg mL−1) against all fungal strains tested with the exception of compounds 2b, 2c, 2d, 2e, 2f, 2g, and 2j against strains O, H (2c only), N (2f only), and E (2g only). All the compounds from series 3 (3a, 3c, 3d, 3e, 3f, and 3g) (i.e., R1 = 5-F, X = N, Y = C; Fig. 1) displayed excellent to good activity (0.06–7.8 μg mL−1) against most of the strains A–P. However, some compounds from series 3 displayed poor activity (15.6–>31.3 μg mL−1). Those include compounds 3a, 3c, 3d, and 3g against strain H, compounds 3e and 3f against strain N, compounds 3e and 3g against strain J, compound 3e against strain P, and all “3”-compounds except 3a against strain O. Among series 3, compounds 3f, 3c, 3a, and 3g were the best in terms of their activity. In the case of monohydrazides from series 4 (i.e., R1 = 5-Br, X = N, Y = C; Fig. 1), compounds 4a, 4c, 4d, and 4f displayed excellent to good activity against strains A–G and I–N (0.24–7.8 μg mL−1). However, compound 4g performed below par with poor activity against strains B, E, G, L, and O. For series 5 (i.e., R1 = 6-F, X = N, Y = C; Fig. 1), compounds 5a, 5c, 5d, and 5f exhibited excellent to good activity (0.06–7.8 μg mL−1) against 14 to 15 (out of 16) of the non-A. fumigatus fungal strains tested, with the exception of compounds 5a, 5c, 5d, and 5f against either strains H, N, and/or O. Compounds 5e and 5g displayed excellent to good activity against strains A–I, K–M, and P (0.98–7.8 μg mL−1), and strains A–G, I, K–N, and P (0.49–7.8 μg mL−1), respectively. Amongst monohydrazides 6a, 6c, 6d, 6f, and 6g (i.e., R1 = 6-Br, X = N, Y = C; Fig. 1), compounds 6a and 6d exhibited excellent to good activity (0.06–7.8 μg mL−1) against all 16 non-A. fumigatus strains tested with the exception of strains H and O. Compounds 6c and 6f displayed excellent to good activity (0.24–7.8 μg mL−1) against all 16 non-A. fumigatus strains tested with the exception of strain O. On the other hand, compound 6g performed poorly against strains B, E, G, H, L, and O. For compounds from series 7 (i.e., R1 = 3,5-diF, X = N, Y = C; Fig. 1), compounds 7a, 7d, and 7f performed better than other members of this series. Compounds 7a, 7d, and 7f exhibited excellent to good activity (0.06–7.8 μg mL−1) against all 16 non-A. fumigatus strains tested, with the exception of compounds 7a, 7d, and 7f against strains H and O, strain H, and strains N and O, respectively. Compounds 7e, and 7g displayed excellent activity (0.49–1.98 μg mL−1) against strains E–G, I, and M, and strains A, G, I, L, M, and P, respectively. For series 8 (i.e., R1 = 3,6-diF, X = N, Y = C; Fig. 1), compound 8d exhibited excellent to good activity (0.24–7.8 μg mL−1) against all the strains tested, with the exception of strain H. Compounds 8a, 8f, and 8g displayed excellent to good activity (0.06–7.8 μg mL−1) against strains A–G, I–N, and P, strains A–M, and P, and strains A–G, I, K–M, and P, respectively. Amongst series 8, compounds 8c and 8e performed subpar when compared to other compounds in the same series. Finally, in the case of series 9 (i.e., R1 = H, X = N, Y = N; Fig. 1), compound 9c exhibited excellent activity (0.49–1.95 μg mL−1) against 14 strains A–G, I–N, and P. Among the remaining compounds in series 9, compounds 9f, 9g, 9b, 9a, 9d, 9e, and 9i displayed excellent to good activity (0.12–7.8 μg mL−1) against all 16 non-A. fumigatus strains tested with the exception of compounds 9f, 9b, 9a, 9d, 9e, and 9i against strains E (9i only was inactive), N (9f only was inactive), and O. Additionally, we explored the activity of compounds 1a, 1d, 1f, 2a–2j, 3a, 3c, 3f, 4a, 4c, 5c, 5d, 5f, 6c, 7a, 7d, 7f, 8a, 8d, 8f, 9a–9c, 9f, and 9g against clinically derived A. fumigatus strains Q, R, and S. A few compounds (2c, 2d, 2e, 5c, and 9c) exhibited good activity against strains Q and R. The highest activity against strain S was 15.6 μg mL−1 for compounds 2d, 2e, 5e and 5f. In summary, compounds 1f, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 3f, 5d, 5f, 8d, 9b, 9c, 9f, and 9g exhibited a comparable or superior activity to that of the FDA-approved antifungal agent AmB against most of the tested strains, with the exception of strains Q, R and S, against which AmB was 4–8-fold more potent than the most active monohydrazides. From all of the observations made for compounds 1a–9j, we concluded that series 2 performed the best followed by series 9.
Based on the promising antifungal activities given in Table 1 and S1,† we selected the entire series 2, and some of the representative compounds from series 1 and 3–9 for further testing against a panel of ten C. auris strains (AR Bank # 381–390) (Table 2 and S2†). Using the same concentration range as above (0.06 to 31.3 μg mL−1) for all of the selected monohydrazides and using AmB as a positive control, we determined MIC values. Monohydrazides 2a, 2b, 2g, 2h, 2i, 5d, 8d, 9b, and 9f displayed excellent activity (0.06–1.95 μg mL−1) against all ten C. auris strains tested. From the remaining set of compounds tested, we found that compounds 2c, 2d, 2e, 2f, 2j, 3c, 3d, 3e, 3g, 4d, 5c, 5e, 5g, 6d, 7d, 7g, 9c, and 9g exhibited excellent to good activity (0.12–7.8 μg mL−1) against all ten C. auris strains tested. Compounds 7f, 8f, and 8g exhibited excellent activity (0.49–1.95 μg mL−1) against all of the strains tested, with the exception of compounds 7f, 8f, and 8g against C. auris strain AR Bank # 390. Similar to the result observed in Table 1, compounds 1b, 1j, 7e, and 8e displayed poor activity against most of the C. auris strains tested.
Fungal strain (AR Bank #) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Cpd # | 381 | 382 | 383 | 384 (K) | 385 | 386 | 387 | 388 | 389 | 390 (L) | 391 | 392 | 393 | 394 | 395 | 396 | 397 | 398 | 399 | 400 |
Strains: 381–390 = Candida auris, 391, 392, and 394 = Candida duobushaemulonii, 393 and 395 = Candida haemulonii, 396 = Kodameae ohmeri, 397 = Candida krusei, 398 = Candida lusitaniae, 399 and 400 = Saccharomyces cerevisiae. | ||||||||||||||||||||
2a | 0.49 | 0.98 | 1.95 | 1.95 | 0.98 | 0.49 | 0.49 | 0.49 | 0.12 | 0.24 | 0.49 | 0.24 | 0.12 | 0.49 | ≤0.06 | 0.24 | 0.24 | 0.98 | 1.95 | 1.95 |
2b | 0.98 | 0.49 | 0.49 | 0.98 | 0.49 | 0.24 | 0.49 | 0.49 | 0.49 | 0.49 | 0.24 | 0.24 | 0.12 | 012 | ≤0.06 | 0.12 | 0.12 | 0.98 | 0.98 | 0.98 |
2c | 3.9 | 0.98 | 0.98 | 0.98 | 3.9 | 0.49 | 0.49 | 3.9 | 0.49 | 0.98 | 1.95 | 1.95 | 0.49 | 0.98 | 0.49 | 0.49 | 0.49 | 1.95 | 7.8 | 7.8 |
2d | 0.98 | 1.95 | 1.95 | 3.9 | 0.98 | 0.49 | 0.98 | 0.98 | 0.49 | 0.98 | 0.49 | 0.49 | 0.24 | 0.24 | 0.12 | 0.24 | 0.24 | 1.95 | 3.9 | 1.95 |
2e | 1.95 | 3.9 | 1.95 | 1.95 | 1.95 | 0.98 | 1.95 | 1.95 | 0.98 | 1.95 | 0.98 | 0.98 | 0.49 | 0.40 | 0.12 | 0.24 | 0.24 | 0.98 | 0.98 | 0.98 |
2f | 1.95 | 1.95 | 1.95 | 3.9 | 0.98 | 0.24 | 0.49 | 0.49 | 0.24 | 0.12 | 0.98 | 0.98 | 0.49 | 0.98 | 0.49 | 0.49 | 0.49 | 0.98 | 1.95 | 0.98 |
2g | 0.49 | 0.49 | 0.49 | 0.49 | 0.49 | 0.49 | 0.12 | 0.12 | 0.24 | 0.12 | 0.12 | 0.49 | 0.49 | 3.9 | 0.24 | 0.98 | ≤0.06 | 1.95 | 0.49 | 0.49 |
2h | 1.95 | 0.24 | 0.49 | 1.95 | 0.49 | 0.12 | 0.49 | 0.49 | 0.49 | 0.98 | 0.49 | 0.24 | 0.98 | 0.24 | 0.24 | 0.12 | 0.24 | 0.49 | 1.95 | 1.95 |
2i | 0.98 | 0.12 | 0.24 | 0.98 | 0.24 | ≤0.06 | 0.24 | 0.24 | 0.24 | 0.49 | 0.24 | 0.49 | 0.49 | 0.24 | 0.24 | 0.12 | 0.12 | 0.49 | 1.95 | 0.98 |
2j | 3.9 | 0.98 | 1.95 | 7.8 | 1.95 | 0.98 | 0.98 | 3.9 | 1.95 | 3.9 | 0.98 | 0.98 | 0.24 | 0.49 | 0.24 | 0.49 | 0.49 | 3.9 | 3.9 | 3.9 |
9b | 0.49 | 0.12 | 0.24 | 0.98 | 0.24 | ≤0.06 | 0.24 | 0.24 | 0.24 | 0.49 | 0.24 | 0.12 | 0.24 | 0.12 | 0.24 | 0.12 | 0.12 | 0.49 | 0.98 | 0.98 |
9c | 1.95 | 0.98 | 1.95 | 1.95 | 7.8 | 0.49 | 0.98 | 3.9 | 0.98 | 0.98 | 3.9 | 0.98 | 0.49 | 1.95 | 0.49 | 0.49 | 0.49 | 1.95 | 7.8 | 3.9 |
9e | 7.8 | 15.6 | 3.9 | 1.95 | 31.3 | 7.8 | 15.6 | 7.8 | 7.8 | 3.9 | 1.95 | 1.95 | 1.95 | 1.95 | 3.9 | 1.95 | 3.9 | 3.9 | 3.9 | 3.9 |
9f | 1.95 | 0.98 | 0.98 | 0.98 | 0.98 | 0.49 | 1.95 | 0.98 | 0.98 | 0.98 | 0.49 | 0.49 | 0.24 | 0.49 | 0.49 | 0.98 | 0.49 | 0.98 | 0.98 | 0.98 |
9g | 3.9 | 1.95 | 0.98 | 3.9 | 0.98 | 0.49 | 0.49 | 0.49 | 0.49 | 0.98 | 0.49 | 0.24 | 0.24 | 7.8 | 1.95 | 0.98 | 1.95 | 3.9 | 7.8 | 1.95 |
AmB | 0.98 | 0.98 | 1.95 | 1.95 | 1.95 | 0.98 | 0.98 | 1.95 | 1.95 | 1.95 | 7.8 | 7.8 | 15.6 | 0.98 | 3.9 | 1.95 | 0.49 | 3.9 | 15.6 | 1.95 |
Next, we tested the compounds from the entire series 2 and selected members of series 9 against a panel of ten additional other fungal strains, including three Candida duobushaemulonii strains (AR Bank # 391, AR Bank # 392, and AR Bank # 394), two Candida haemulonii strains (AR Bank # 393 and AR Bank # 395), two Saccharomyces cerevisiae strains (AR Bank # 399 and AR Bank # 400), and one each of the following strains: Kodameae ohmeri (AR Bank # 396), Candida krusei (AR Bank # 397), and Candida lusitaniae (AR Bank # 398) (Table 2). Monohydrazides 2a, 2b, 2e, 2f, 2h, 2i, 9b, and 9f displayed excellent activity (0.06–1.95 μg mL−1) against all ten additional strains tested, whereas the remaining compounds 2c, 2d, 2g, 2j, 9c, 9e, and 9g exhibited excellent to good activity (0.06–7.8 μg mL−1) against all ten strains. Overall, as shown in Table 2 and S2,† the most active monohydrazides displayed excellent activity against a panel of ten C. auris (AR Bank # 381–390) and ten other fungal strains (AR Bank # 391–400).
Next, we looked into the effect of varying the R1 substituent while keeping R2 constant (i.e., comparing all “a” compounds 1a–9a, then all “b” compounds 1b–9b, etc.). We found that the monohydrazides displaying the best antifungal activity generally had H (series 2 where X = N and Y = C as well as series 9 where X = N and Y = N) as an R1 substituent. For compounds with R2 = 3-F (a), the most active compounds (from most to least active) were 2a (R1 = H, X = N, Y = C), 9a (R1 = H, X = N, Y = N), 7a (R1 = 3,5-diF), and 8a (R1 = 3,6-diF), respectively. For compounds with R2 = 3-Cl (b), the introduction of H as an R1 substituent resulted in compounds 2b and 9b with a better overall antifungal activity. The most active compounds amongst monohydrazides with R2 = 3-OMe (c), compounds 9c, 2c, 3c, and 5c, had H (X = N, Y = C), H (X = N, Y = N), 5-F, and 6-F as R1 substituents, respectively. For compounds with R2 = 4-F (d), the most active compounds 2d, 8d, 5d, and 9d possessed H (X = N, Y = C), 3,6-diF, 6-F, and H (X = N, Y = N) as R1 substituents, respectively. For compounds with R2 = 4-Cl (e), the most active compounds 2e and 9e possessed H as R1 substituents. For compounds with R2 = 4-OMe (f), the most active compounds had 6-F (5f), H (2f and 9f), 2,4-diF (1f), and 5-F (3f) as R1 substituents. For monohydrazides with R2 = 2,4-diF (g), the presence of H and 5-F as R1 substituents resulted in compounds 9g, 2g, and 3g with better overall antifungal activity than those with other R1 substituents. Similar activity profiles were observed for compounds with R2 = 2,5-diF (h), R2 = 3,5-diF (i), and R2 = 3,5-diCl (j). The introduction of H (X = N, Y = C) and H (X = N, Y = N) as R1 substituents resulted in compounds 2h, 9h, 2i, 9i, 2j, and 9j with better overall activity. In general, we observed that the monohydrazides with the best overall antifungal activity with diverse R2 groups had H (series 2 and 9) as an R1 substituent.
For further in-depth analysis of the antifungal activity, we explored the effect of regioisomers on ring A by comparing series 3 (R1 = 5-F) with series 5 (R1 = 6-F) and series 4 (R1 = 5-Br) with series 6 (R1 = 6-Br). We observed that compounds 5d (R2 = 4-F), 5e (R2 = 4-Cl), and 5f (R2 = 4-OMe) performed better than their counterparts 3d (R2 = 4-F), 3e (R2 = 4-Cl), and 3f (R2 = 4-OMe), whereas compounds 3a (R2 = 3-F) and 3g (R2 = 2,4-diF) displayed better activity compared to 5a (R2 = 3-F) and 5g (R2 = 2,4-diF). While comparing series 4 (R1 = 5-Br) with series 6 (R1 = 6-Br), we found compounds 6d (R2 = 4-F), 6f (R2 = 4-OMe), and 6g (R2 = 2,4-diF) to be better antifungals than their counterparts 4d (R2 = 4-F), 4f (R2 = 4-OMe), and 4g (R2 = 2,4-diF). From the data reported above, we were able to point to the superiority of series 5 and 6 (R1 = 6-F and R1 = 6-Br) over series 3 and 4 (R1 = 5-F and R1 = 5-Br). For disubstituted monohydrazides (series 7 and 8), compounds 7a (R2 = 3-F) and 7f (R2 = 4-OMe) exhibited similar activity to 8a (R2 = 3-F) and 8f (R2 = 4-OMe), whereas compounds 8c (R2 = 3-OMe), 8d (R2 = 4-F), and 8g (R2 = 2,4-diF) were better than their counterparts from series 7. These observations point to fact that having a substituent at the 6-position of ring A substantially increases the activity of the compounds.
We next evaluated the impact of a halogen identity on antifungal activity by comparing series 3 and 4 (5-F vs. 5-Br) as well as series 5 and 6 (6-F vs. 6-Br). For every R2 substituents, compounds from series 3 (3a, 3c, 3d, 3f, and 3g) performed significantly better than compounds from series 4 (4a, 4c, 4d, 4f, and 4g). A similar trend was observed in the case of series 5 and 6. Compounds 5a (R2 = 3-F), 5c (R2 = 3-OMe), 5d (R2 = 4-F), 5f (R2 = 4-OMe), and 5g (R2 = 2,4-diF) performed better than their counterparts 6a (R2 = 3-F), 6c (R2 = 3-OMe), 6d (R2 = 4-F), 6f (R2 = 4-OMe), and 6g (R2 = 2,4-diF). From these observations, we concluded that a fluorine is preferred over a bromine as a R1 substituent for antifungal activity.
Finally, we explored the effect of the position of the R2 substituents on ring B (i.e., 3- vs. 4-position) within each series for the entire nine series of compounds (1avs.1d, 1cvs.1f, and 1bvs.1e, etc.). For monohydrazides where R2 substituents are halogens (3-F (a) and 4-F (d) or 3-Cl (b) and 4-Cl (e)), in general we observed better antifungal activity for 3-position isomers over 4-position isomers. Indeed, for compounds with R2 = 3-F (a) and 4-F (d), 1a, 2a, 3a, 4a, and 6a performed better than their counter parts 1d, 2d, 3d, 4d, and 6d. Similarly, for compounds with R2 = 3-Cl (b) and 4-Cl (e), 1b, 2b, and 9b displayed better antifungal activity than compounds 1e, 2e, and 9e. However, for compounds with R2 = OMe (1c–9c with 3-OMe vs.1f–9f with 4-OMe), the 4-postion isomers were better antifungals when compared to the 3-position isomers. Compounds 1f, 3f, 5f, 7f, and 8f displayed better activity than their counter parts 1c, 3c, 5c, 7c, and 8c. Next, we explored the effect of a halogen identity on ring B (i.e., F (a and d) vs. Cl (b and e)) on antifungal activity. When comparing compounds with R2 = 4-F (d) and R2 = 4-Cl (e), we found that the presence of a fluorine (compounds 1d, 3d, 5d, 7d, 8d, and 9d) is preferred over that of a chlorine atom (1e, 3e, 5e, 7e, 8e, and 9e). However, when comparing compounds with R2 = 3-F and R2 = 3-Cl (1avs.1b, 2avs.2b, and 9avs.9b), we did not observe any distinct differences in antifungal activity.
Fig. 3 2D bar graphs normalized at 100% depicting the dose-dependent cytotoxic activity of monohydrazides 2a, 2b, 2d, 2g, 2i, 9a, 9b, 9c, 9f, and 9g, as well as AmB and VRC against A. J774A.1, B. HEK-293, and C. HepG2 mammalian cell lines. Note: For Triton X-100® (TX) the eight bars are colored differently and correspond to the colors of the respective compounds for which TX was used as a positive control. Note: The corresponding non-normalized data are presented in Fig. S173.† |
We also investigated in silico the metabolic properties of potent compounds 2a–2j, 9a–9c, 9f, and 9g using SwissADME against the five isoforms of cytochrome P450 (CYP) monooxygenase (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4). None of the compounds (with the exception of compound 2j against CYP2C19 and CYP2D6) were identified as possible inhibitors of CYP2C19, CYP2C9, CYP2D6, and CYP3A4. However, except for compound 2i, all compounds were predicted to inhibit CYP1A2 (Table S6†).
Fig. 4 Time-kill curves for C. albicans ATCC 10231 (strain A) with AmB (0.98 μg mL−1, purple), 2b (1× MIC, fushia), 2b (4× MIC, pale pink), and untreated (grey). |
Fig. 6 Graph showing the MIC values over 15 passages for compounds 2a (turquoise circles) and 2b (fushia inverted triangles) against C. albicans ATCC 10231 (strain A). |
ADMET | Absorption, distribution, metabolism, excretion, toxicity |
AmB | Amphotericin B |
ATCC | American Type Culture Collection |
BBB | Blood–brain barrier |
CFG | Caspofungin |
CFU | Colony-forming unit |
CL | Clearance |
FLC | Fluconazole |
hERG | Human ether-à-go-go-related |
HIA | Human intestinal absorption |
IFIs | Invasive fungal infections |
MIC | Minimum inhibitory concentration |
Pgp | P-glycoprotein |
PPB | Plasma protein binding |
SAR | Structure–activity relationship |
TPSA | Total polar surface arear |
TX | Triton X-100® |
VD | Volume distribution |
VRC | Voriconazole |
Footnotes |
† Electronic supplementary information (ESI) available: Experimental procedures for the preparation and characterization of compounds 1a–9j as well as MIC value determination by in vitro antifungal, in vitro cytotoxicity, time-kill, biofilm disruption, prevention of biofilm formation, and development of resistance assays. 1H and 13C NMR spectra, HPLC traces, and HR-MS figures for compounds 1a–9j. Tables for experimentally determined MIC values as well as in-silico predictions of physicochemical and ADMET properties. See DOI: https://doi.org/10.1039/d3md00167a |
‡ These two authors contributed equally to this work. |
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