N-1 ester modification and their anti-trypanosomal effect

In Table 15, a linear relationship between logP and the starting rhodanine derivatives was observed. Following this trend, elongated N-1-sidechain rhodanines showed similar activities to ester derivatives due to similar logP values. However, a methyl ester did not follow this trend;

although the logP (0.46) was relatively low, its activity was better than compounds with higher logPs (0.82, 0.84). Thus far, it has been established that elongated derivatives with a free carboxylic acid had activities against T. brucei in the range of 9.0-72.8 µM. In this chapter, ester derivatives of rhodanine-N-acetic acid were screened for their trypanocidal activity (Table 17).

Hydroxy-modified ester analogues

The most potent compound against T. brucei growth was the catechol modified ethyl ester 14t (Table 17). 14t had an activity of GI₅₀ 1.6 µM against T. brucei, without showing any

Table 17: Anti-trypanosomal activity of ester analogues and their toxicity, sorted by activity and toxicity.

N S S

O R¹O

O

R² R³ R⁴ R⁵

GI₅₀[µM]

# R¹ R² R³ R⁴ R⁵ T. brucei SI (T. brucei) T. cruzi HL60

14t Et H OH OH H 1.6 ± 0.1 >62.5 n.a. >100

14o Et H H tBu H 1.7 ± 0.1 >59 >100 >100

14v Et H OBn H H 4.4 ± 1.1 >23 >100 >100

14j tBu H H H H 9.6 ± 0.8 >10 >100 >100

14a Et H CH3 H H 1.5 ± 0.3 21 >100 31.0 ± 0.8

14b tBu H CH₃ H H 1.8 ± 0.1 14 5.1 ± 0.5 25.0 ± 1.6

14g Et CH₃ H H H 1.3 ± 0.1 10 >100 12.7 ± 0.9

14c Me H CH3 H H 1.3 ± 0.1 9 >100 12.0 ± 1.7

14h tBu CH₃ H H H 5.2 ± 0.3 9 >100 48.2 ± 7.0

14d Et H H CF₃ H 1.4 ± 0.1 8 n.a. 11.8 ± 0.5

14e Me H H CF3 H 1.4 ± 0.1 7 17.4 ± 1.1 9.9 ± 1.8

14k Et CF₃ H H H 1.4 ± 0.1 7 n.a. 9.3 ± 3.7

14l Et H CF₃ H H 1.7 ± 0.1 7 n.a. 11.3 ± 3.7

14m Et CF3 H H CF3 1.8 ± 0.1 n.a. n.a. n.a.

14w Et H H OBn H 17.0 ± 0.9 6 >100 >100

14i Et H H H H 8.3 ± 0.7 5 94.6 ± 1.8 38.0 ± 1.8

14y Et H OMe OH OMe 11.7 ± 1.8 3 46.6 ± 0.5 38.1 ± 6.4

14p Et OH H H H 1.7 ± 0.1 2 n.a. 4.1 ± 0.7

14s Me H OH OH H 11.1 ± 0.1 2 n.a. 23.6 ± 1.1

14n Et H H CH3 H 15.6 ± 1.4 2 n.a. 29.3 ± 8.3

14q Et H OH H H 4.8 ± 0.9 2 n.a. 9.9 ± 0.5

14f tBu H OBn OBn H >100 n.a. >100 n.a.

14x Et H OBn OBn H >100 >1 >100 >100

14u Et OH H OH H 1.7 ± 0.1 n.a. 4.7 ± 0.6 n.a.

35b Me H OH OH H 10.1 ± 1.6 n.a. n.a. n.a.

14r Et H H OH H 17.6 ± 0.1 n.a. >100 n.a.

3 Rhodanine-N-acetic acid derivatives

Scheme 21: Catechol reactivity to react with biological nucleophiles or cross-link proteins, adapted scheme by Stanwell et. al^[198].

toxicity against HL60 cells at 100 µM. This was a surprising observation, since the analogous catechol modified free acid2u did not show any trypanocidal activity. The simple esterification resulted in the transformation of an inactive compound against T. brucei growth to one of the most active inhibitors both in terms of anti-trypanosomal activity and selectivity against HL60 cells (SI >63, Table 17). In particular the missing toxicity against HL60 cells was significant, since similar compounds, such as the N-1-unmodified catechol rhodanine36a (Table 41, page 152) has previously been reported as potent inhibitor of HL60 cell growth.^[197] Compound 37 has been shown to inhibit oncoprotein aggregation of c-Myc Max, therefore preventing DNA binding and leading to apoptosis in HL60 cells.^[197]Indeed,36a showed a GI₅₀value of 45 µM against HL60 cells growth, confirming the previously reported IC₅₀of 23 µM.^[197]The additional substituent in position N-1 in compound14t led to complete loss of toxicity against HL60 cell growth, potentially showing that c-Myc Max aggregation was not inhibited. However, the ethyl ester modification was essential for retaining the selectivity as the corresponding methyl ester 14s had only a selectivity index (SI) of 2 and displayed a ten-fold reduction in anti-parasitic activity (GI50 11.1 µM). This indicates the potential problem of catechol-containing molecules in medicinal chemistry.^[198] The cytotoxicity of catechol derivatives is believed to derive from oxidation of the hydroxyl groups to quinones, which can react with biological nucleophiles such as glutathione or cross-link proteins (Scheme 21).^[198,199]

However, this mode-of-action cannot be generalised, as the free acid of14t has previously been reported as non-covalent ("affinity-based") probe for the NAD(P)H site of dehydroge-nases.^[107,108] Indeed, similar catechol derivatives showed nM activity against the NAD(P)H-dependant enoyl acyl carrier protein reductase of Plasmodium falciparum (PfENR).^[112] The catechol moiety has been found to be essential for PfENR inhibition and thus for type II fatty acid biosynthesis inhibition.^[112]T. brucei uses a related elongase pathway for de novo synthe-sis of myristate, an essential building block for GPI anchor biosynthesynthe-sis,^[200] (further details in section 5.1) possibly explaining the low µM anti-trypanosomal activity.

A third possible mode of action was derived during the evaluation of the activity and toxicity assays against T. brucei and HL60 cells. Stock solutions of catechol derivatives14t, 14s, and

35b in DMSO are distinguished by their bright yellow-orange colour. However, in aqueous solutions these compounds were bright red. Catechol rhodanine 36a has previously been found to form brown complexes with Fe(III) ions.^[201] In addition, Mn(II) and Mg(II) formed purple-red complexes with catechol rhodanine under basic conditions.^[201] It seemed evident that14t, 14s, and 35b formed metal-complexes in the assay medium. Although the red colour might have indicated Mn(II) or Mg(II) chelation, the neutral pH of the assay medium made Fe(III) more likely.

Other hydroxy-substituted methyl and ethyl ester derivatives (14p, 14s, 14q, 14u, 35b, 14r, and 14y) showed good activity against T. brucei growth (GI₅₀ 1.7-17.6 µM), however the selectivity indices were less preferable for derivatives14p, 14s, and 14q (SI 2). A particularly interesting inhibitor was14u, as it showed activity in the lower µM range for both T. brucei and T. cruzi (GI₅₀1.7 and 4.7 µM). The previously mentioned formation of bright red colours in the assay medium interfered with the MTT formazan readout, therefore derivatives14p, 35b, and 14r could not be assessed against T. cruzi, although the MIC value was 100 µM.

Methyl-, trifluoromethyl- and tert-butyl-substituted benzylidene derivatives and their trypanocidal activity

Methyl- and trifluoromethyl-substituted ester derivatives 14a–l and 14n had low µM activity against T. brucei with GI₅₀values ranging from 1.3-15.6 µM and had SI of 2-21. These deriva-tives showed general cytotoxic effects against T. brucei and HL60 cells. Different ester mod-ifications (methyl, ethyl or tert-butyl) had no effect on the activity against T. brucei or toxicity against HL60 cells. However, modification of the ester moieties resulted in an increased activ-ity against T. cruzi. The ethyl ester derivative14a did not show any trypanocidal activity against T. cruzi at 100 µM, while the methyl ester substitution increased activity by a factor of 20 (GI50

5.1 µM). Modification of the ester group also proved beneficial for the unsubstituted benzyli-dene derivatives14i and 14j. The ethyl ester modification in 14i increased activity against T.

brucei by a factor of 7 (GI₅₀8.3 µM) compared to the free acid analogue2a (GI₅₀56.0 µM), but 14i also showed increased toxicity against HL60 cells (GI₅₀ 38 µM). Substitution of the ethyl to a bulky tert-butyl ester had no effect on the anti-trypansomal activity against T. brucei, but has proven beneficial in terms of its toxicity. The tert-butyl ester derivative14j retained activity at GI₅₀ 9.6 µM and did not show any toxicity against HL60 cells at 100 µM. A similar conclu-sion could be drawn by comparing the 5-benzylidene modifications in para position. The bulky lipophilic trifluoromethyl derivatives 14d and 14e displayed low µM activity against T. brucei, but also demonstrated significant toxicity against HL60 cells (SI 7). Bio-isosteric replacement of the trifluoromethyl group (Taft E value -2.4), to the only slightly larger tert-butyl group (Taft E value -2.78) resulted in complete loss of toxicity at 100 µM against HL60 cells, while activity against T. brucei was retained at GI₅₀ 1.7 µM.^[193] The retention in activity might suggest that the trifluoromethyl group served as a lipophilic substituent in para-position of the 5-benzylidene

3 Rhodanine-N-acetic acid derivatives

moiety.

Benzyloxy-modified 5-benzylidene ester derivatives and their anti-trypanosomal activity Benzyloxy-modified rhodanine-N-acetic acid derivatives2b, 2c, and 2d were of particular inter-est, as they showed promising activity against DPMS (residual activity 10-23 %), an essential enzyme in the GPI-anchor biosynthesis.^[68] It was pleasing to observe, that the 3-benzyloxy-modified ethyl ester 14v showed low µM activity against T. brucei (GI₅₀ 4.4 µM) and most importantly did not reveal any toxicity against HL60 cells at 100 µM. These results might in-dicate a correlation between the in vitro anti-trypanosomal activity against T. brucei and the enzymatic activity of DPMS. The ester modification could facilitate membrane diffusion, allow-ing a higher concentration of the inhibitor to reach the enzymatic site for effective inhibition.

Interestingly the 4-benzyloxy-ethyl ester was four times less active in vitro against T. brucei, although it showed two times better DPMS inhibition (Table 13, 10 % residual activity) than 14v (23 % residual activity).^[68] However, it is not known if the active inhibitor of DPMS is the free acid or its ester analogue. Moreover, potentially multiple targets in T. brucei could be affected by the modified analogues 14v and 14w. The 3,4-bis-benzyloxy ester modified analogues14f and 14x did not demonstrate any trypanosomal activity at 100 µM. These ana-logues might have been too lipophilic to pass through the parasitic plasma membrane and therefore demonstrate the limits of increasing lipophilicity to gain anti-trypanosomal activity.

3.3.7 Compounds with improved anti-parasitic activity identified after first generation