Further optimisation of structure classes A,C and D

In the next round of inhibitor optimisation, the free carboxylic acid of the elongated analogues was masked as its ethyl ester. This modification resulted in low µM active compounds for the simple N-acetic ester modified series. The thiocarbonyl in the rhodanine was replaced by a carbonyl group. A recent review about rhodanine derivatives suggested that this thio-carbonyl group is a major contributor to the ubiquitous binding of rhodanine derivatives.^[83]

The interactions of the thiocarbonyl group in rhodanine derivatives in protein X-ray structures in the PDB database has been analysed and revealed that this group undergoes a significant amount of polar and intermolecular interactions.^[83]Therefore, replacement of this thiocarbonyl might increase selectivity towards a particular target, possibly simultaneously reducing toxic-ity against HL60 cells. The modifications on the 5-benzylidene moiety were chosen based on previous results. Hydroxy, methyl and trifluoromethyl substituents resulted in low µM try-panocidal compounds, if combined with an ester moiety on the N1-side of the rhodanine core structure (Table 17). The results of the screening of these inhibitors against T. brucei, T. cruzi and HL60 cells is summarised in Table 19. The entries in the table were sorted by increas-ing activity against T. brucei and decreasincreas-ing toxicity against HL60 cells. Not surprisincreas-ingly, the top entries of Table 19 constituted catechol derivatives (7k, 15f, and 16i). Substitution of the thiocarbonyl did not effect activity against T. brucei. The dione derivative 7k had a GI50of 1.4 µM against T. brucei, whilst showing no toxicity against HL60 cells. Alkylation of the 3,4-dihydroxy motif with benzyl groups totally abolished anti-trypanosomal activity. This might indicate that the catechol moiety could be involved in hydrogen bonding interactions.

Alterna-3 Rhodanine-N-acetic acid derivatives

Table 19: 3rd generation optimisation of compound classes A, C and D.

N S X

O O

O

R¹ R² R³ R⁴ n

GI₅₀[µM]

# X n R¹ R² R³ R⁴ T. brucei SI T. brucei T. cruzi HL60

7k O 1 H OH OH H 1.4 ± 0.2 >71 >100 >100

15f S 2 H OH OH H 0.9 ± 0.1 35 n.a. 31.2 ± 2.8

16i S 3 H OH OH H 1.4 ± 0.3 21 n.a. 29.0 ± 4.2

16a S 3 H H H H 7.4 ± 5.2 >14 n.a. >100

16e S 3 CF₃ H H CF₃ 9.5 ± 0.1 5 >100 47.9 ± 4.6

15a S 2 H H H H 4.5 ± 0.3 4 >100 16.5 ± 1.2

16f S 3 OH H H H 1.3 ± 0.1 n.a. 10.9 ± 0.1 n.a.

15c S 2 H CH₃ H H 10.2 ± 0.2 2 >100 17.6 ± 2.5

15b S 2 CH₃ H H H 10.6 ± 0.3 3 >100 35.6 ± 2.9

16b S 3 CH₃ H H H 11.5 ± 0.1 4 >100 40.5 ± 3.3

16c S 3 H CH₃ H H 12.0 ± 1.1 3 >100 31.5 ± 1.6

7e O 1 CF₃ H H H 13.0 ± 1.2 >8 >100 >100

15d S 2 H H CH₃ H 13.1 ± 2.4 3 >100 44.4 ± 5.6

7f O 1 H CF₃ H H 13.3 ± 0.4 >8 >100 >100

7c O 1 H CH₃ H H 14.7 ± 1.7 >7 >100 >100

16d S 3 H H CH₃ H 15.1 ± 2.2 >7 >100 >100

7b O 1 CH₃ H H H 15.2 ± 2.3 >7 >100 >100

7d O 1 H H CH₃ H 16.3 ± 1.8 >6 >100 >100

16g S 3 H OH H H 16.8 ± 1.0 21.4 ± 4.2 n.a.

15e S 2 H H OH H 17.4 ± 0.5 1 >100 9.2 ± 2.0

7j O 1 H OBn OBn H >100 >1 n.a. >100

tively the 3,4-benzyloxy substituent may be too bulky to be accommodated in the active site, or it may have been too lipophilic to pass through the parasite membrane. The elongation of the side-chain in N-1 position in the rhodanine moiety combined with the ester modification and a catechol motif in position five resulted in the first inhibitor against T. brucei growth in the sub-µM range. The elongated analogue15f had a GI50 value of 0.9 µM and displayed a SI of 35 towards HL60 cells. Increasing the methylene linker even further, as shown in compound 16i did not improve activity against T. brucei. Interestingly, the combination of an elongated side chain (n=3) combined with a hydroxy substituent on the 5-benzylidene moiety resulted in compounds 16f and 16g, which displayed moderate activities against T. cruzi (GI₅₀ 10.9-21.4 µM). However, the shorter (n=2) linker analogue did not show any activity against T. cruzi, but toxicity in the lower µM range against HL60 (GI₅₀9.2 µM).

Applying the elongation and esterification strategy on methyl or trifluoromethyl benzylidene modified rhodanine derivatives did not improve their anti-parasitic activity (GI₅₀9.5-16.3 µM) or toxicity against HL60 cells (SI 2-8) compared to derivatives 14a–l. The substitution of the thiocarbonyl to a carbonyl group improved their toxicity profile and none of the inhibitors displayed any toxicity against HL60 cells at 100 µM. But the replacement also reduced the anti-parasitic activity by a factor of 10 (GI₅₀ 13.0-16.3 µM).

Optimisation of compound class B

In the first screening of rhodanine derivatives, a 5-(benzylidene-3 benzyloxy) rhodanine-N-acetic acid derivative (2c, Table 20) has been identified as DPMS inhibitor of T. brucei, but did not display any activity against T. brucei in vitro.^[68] The introduction of an ethyl ester moiety as replacement for the free acid resulted in low µM activity against T. brucei (14v, Table 20).

In order to improve the anti-trypanosomal activity, various substituents on the 3-benzyloxy moiety were introduced. Furthermore, the side-chain linker was elongated by an additional methylene-unit, and the thiocarbonyl was substituted with a carbonyl group. Lastly prolonged N-1-side-chain analogues with a free acid moiety were compared to ester analogues. The assessed inhibitors are displayed in increasing order of activity against T. brucei and decreas-ing toxicity against HL60 cells (Table 20). Indeed, substitution of the 3-benzyloxy substituent improved activity against T. brucei by a factor of 3 compared to the unsubstituted analogue 14v. The inhibitor 19c displayed low µM activity against T. brucei (GI₅₀ 1.5 µM) and only mi-nor toxicity against HL60 cells (SI 59). The derivative 14v is equipped with a para-methoxy substituent on the 3-benzyloxy-moiety. Using this modification and replacing the thiocarbonyl to a carbonyl group furnished 19e, revealing a 10-fold decrease in activity against T. brucei.

Similarly, increasing the length of the N-1 side-linker in compound19e caused a 10-fold drop in anti-trypanosomal activity against T. brucei, this is similar to previous reports that substitu-tion to the carbonyl group results in loss of activity.^[81] It was interesting to observe that the starting para-methoxy substituted aldehyde18c did not display any anti-trypanosomal activity

3 Rhodanine-N-acetic acid derivatives

Table 20: Optimisation of compounds from class B, substitution of the 3-benzyloxy-substituent.

at 100 µM. While the analogous meta-methoxy substituted aldehyde18b had a moderate ef-fect on trypanosomal growth (GI₅₀15.5 µM). However, the corresponding condensation product 19a was 10-fold less active than the para-substituted analogue 19c.

The introduction of a pyridine-moiety in compounds19k, 19j, and 19l resulted in low µM ac-tive inhibitors of T. brucei growth (GI501.7 µM). But these derivatives displayed less selectivity between T. brucei and HL60 cells (SI 7-9), compared to the methoxy substituted derivative 19c (SI 59). Similar to the methoxy-substituted analogues, the meta-pyridine substituted alde-hyde 18e showed moderate anti-trypanosomal activity while the ortho- and para-substituted analogues did not display any activity against T. brucei at 100 µM. The combination of a free carboxylic acid and an elongated side-chain with the pyridine-substiution furnished com-pounds19h, 19g, and 19f, which displayed only moderate activity against T. brucei (GI₅₀ 60.7-68.3 µM).

Compound 34 was a protein-reactive photo-labelling probe. This probe was designed to bind to its target protein via an aryl-azide moiety, while a second photo-stable aliphatic azide serves as a tag for biotin or fluorophorores (such as AlexaFluor 488). It was pleasing to observe that although relatively large azide substituents were introduced, the probe 34 retained most of its anti-trypanosomal activity (GI₅₀ 12.9 µM) compared to the original lead structure14v (GI₅₀4.4 µM).

In order to study the importance of the oxygen-linker in the benzyloxy-substituted ana-logues, the heterocycle26a was assessed for its anti-trypanosomal potential. The 5-(benzy-lidene-3-benzyloxy) moiety has been replaced with a 2-amino(benzylidene)-pyridine group in compound26a. This derivative was the first inhibitor in this series to display a trypanocidal effect both in T. brucei and T. cruzi, however the activity against T. cruzi might correlate to the toxicity towards HL60 cells. Yet this inhibitor was a good starting point for the development for more potent and in particular less toxic inhibitors of T. brucei and T. cruzi growth.

3.3.9 Heterocyclic modifications on position 5 of N-1-substituted rhodanine and thiazolidine-2,4-dione derivatives

Heterocyclic analogues, such as the rhodanine-N-acetic ester derivative26a (Table 20) has been identified as low µM growth inhibitor of T. brucei and T. cruzi, but also displayed high tox-icity against HL60 cells (GI50 10.0 µM). In this chapter, several 5-pyridine modifications have been assessed for their anti-trypanosomal activity (Table 21). The most active inhibitor in this series had a 3-pyridinyl modification on the 5-position of the rhodanine-N-acetic ester moiety.

Compounds26g and 26f displayed anti-trypanosomal activity against T. brucei in the low µM range (GI₅₀1.5 µM) and against T. cruzi at 100 µM (MIC 100 µM). The exact GI₅₀values could not be obtained due to interference of the derivatives with the viability dye MTT used in the growth inhibition assay for T. cruzi. Furthermore, both inhibitors 26g and 26f possessed a

3 Rhodanine-N-acetic acid derivatives

Table 21: Heterocyclic modified rhodanine-N-acetic acid and there ester analogues.

N S X

O R¹O

O

R²

GI₅₀[µM]

# x R¹ R² T. brucei SI T. brucei T. cruzi HL60

26g S Et 3-pyridinyl 1.5 ± 0.2 34 n.a. 51.5 ± 2.5

26f S Et 2-pyridinyl 1.5 ± 0.3 >67 n.a. >100

21a S Et 2-cl-4-pyridinyl 1.7 ± 0.1 n.a. 6.9 ± 0.6 n.a.

26c S H 3-pyridinyl 116.0 ± 6.4 >1 >100 >100

26i O Et 4-pyridinyl 16.4 ± 1.7 >6 n.a. >100

26b S H 2-pyridinyl >100 >1 >100 >100

26h S Et 4-pyridinyl >100 >1 >100 >100

26d S H 4-pyridinyl >100 >1 >100 >100

26j S Et 2-furanyl 7.4 ± 2.1 7 n.a. 48.1 ± 2.4

good toxicology profile (SI 34-(>67)). The anti-parasitic effect against T. brucei could already be observed in the free carboxylic acid derivative26c, which inhibited growth of T. brucei at 100 µM by 55 % (data not shown) and resulted in a GI₅₀116 µM. The transformation of the acid to the ethyl ester resulted in a compound with low µM activity and a good selectivity profile.

A 4-pyridinyl-modification on position 5 of the rhodanine-N-acetic acid (26d) and ester (26h) resulted in a complete loss of anti-trypanosomal activity against both T. brucei and T.

cruzi. However, substituting the thiocarbonyl to a carbonyl-group in compound 26i restored some of the anti-trypanosomal activity against T. brucei (GI50 16.4 um). In addition the intro-duction of a 2-chloro-substituent next to 4-pyridinyl-moiety restored activity. Indeed, compound 21a was equipotent against T. brucei and T. cruzi as the 2- and 3-pyridinyl substituted inhibitors 26g and 26f (GI₅₀1.5 µM).