Spirocarbocyclic and aryl-substituted 2,6-diketopiperazino-1-acetohydroxamic and spirocarbocyclic N-[(2,6-diketopiperazin-1-yl)acetyl]glycinehydroxamic acids: design, synthesis and evaluation of their in vitro antitrypanosomal activity."

Doctoral Dissertation uoadl:1309769 508 Read counter

Unit:
Τομέας ΦΑΡΜΑΚΕΥΤΙΚΗΣ ΧΗΜΕΙΑΣ
Library of the School of Science
Deposit date:
2016-10-21
Year:
2016
Author:
Τσατσαρώνη Αλεξάνδρα
Dissertation committee:
Ανδρέας Τσοτίνης Καθηγητής
Original Title:
“Σπειροκαρβοκυκλικά και αρυλοϋποκατεστημενα 2,6-δικετοπιπεραζινο-1-ακετοϋδροξαμικά και σπειροκαρβοκυκλικά Ν-[(2,6-δικετοπιπεραζιν-1-υλ)ακετυλο]γλυκινοϋδροξαμικά οξέα: Σχεδιασμός, Σύνθεση και Αξιολόγηση της αντιτρυπανοσωμιακής in-vitro δράσης”
Languages:
Greek
Translated title:
Spirocarbocyclic and aryl-substituted 2,6-diketopiperazino-1-acetohydroxamic and spirocarbocyclic N-[(2,6-diketopiperazin-1-yl)acetyl]glycinehydroxamic acids: design, synthesis and evaluation of their in vitro antitrypanosomal activity."
Summary:
The present PhD thesis refers to the design, synthesis and evaluation of the in
vitro anti-trypanosomal activity of new spirocarbocyclic (adamantano,
cyclooctano and cycloheptano derivatives),
2,6-diketopiperazino-1-acetohydroxamic acids (compounds 1-6),
3-alkyl-3-aryl-2,6-diketopiparazino-1- acetohydroxamic acids (compounds 7-18)
and spirocarbocyclic N-[(2,6-diketopiperazin-1-yl)glycine hydroxamic acids
(compounds 19-29).
The rationale behind the design of compounds 1-18, was based on our previous
findings according to which the introduction of the acetohydroxamic moiety
(CH2CONHOH) on the imidic nitrogen atom of lipophilic spirocarbocyclic
2,6-diketopiperazine scaffolds, leads to very potent trypanocidals (IC50
6.8-1870 nΜ, compounds VIIIa-e, IX, Xa-d, XΙa and XIb, Table 1).
Analogues 1-4 were synthesized in order to investigate whether the structural
features of the substituent on the methylenic carbon, near the basic nitrogen
atom of the 2,6-diketopiperazino ring, have an effect on the antitrypanosomal
activity.
Moreover, the synthesis of the N-methylhydroxamic acids (2o hydroxamic acids 5
and 6) aimed at probing the influence of the methyl substitution on the
conformation of the carbohydroxamic group and hence the antitrypanosomal
activity of the spirocarbocyclic 2,6-diketopiperazino-1-acetohydroxamic acids.
The structural modification, which involves the replacement of the spiro-linked
carbocyclic ring by benzene (substituted and unsubstituted) and an alkyl
substituent (CH3, (CH2)2CH3, (CH2)3CH3), led to the design of compounds 7-18.
In a previous publication we demonstrated that the presence of the
acetohydroxamic group in compounds VIIIa-e, IX, Xa-d, XΙa and XIb constitutes a
prerequisite for trypanocidal activity, which was linked to the ability of
these molecules to inhibit a metalloenzyme, by trapping the metal ion on its
active centre. Thus, in order to augment the formation of a
carbohydroxamic-enzymic metal ion complex, the acetohydroxamic moiety
(CH2CONHOH) was replaced by an N-acetoglycine hydroxamic residue
(CH2CON(R)CH2CONHOH). The introduction of this functionality was hoped to lead
to an enhanced complex stabilisation, due to additional interactions between
the acetamido portion (CH2CONR) and the biological target(s) in the active
centre’s nearby sites. Based on this assumption compounds 19-29 were designed
and synthesised.
The carboxylic acids 43-45 (Scheme 4), 59, 60 (Scheme 5), 83-86 (Scheme 8),
107-110 (Scheme 9), 124 and 125 (Scheme 10), are precursors of the target
molecules 1, 3-6, 7, 8, 10-13, 15-18 and were synthesised by following the
appropriate methodologies we have previously developed. Thus, the coupling of
the aforementioned carboxylic acids with O-benzylhydroxylamine or
O-benzyl-N-methylhydroxylamine, in the presence of 1,1’-carbonyldiimidazole
(CDI), leads to to the O-benzylcarbohydroxamic derivatives 46, 48, 49, 61, 62,
87, 88, 90, 91, 111, 112, 114, 115, 126 and 127. Hydrogenolysis of their benzyl
group gives the acetohydroxamic acids 1, 3-6, 7, 8, 10-13, 15-18. The synthesis
of the acetohydroxamic acids 2, 9 and 14, involved the treatment of the
respective 4-methoxybenzyl esters 40, 80 and 104 with TFA in DCM, and amidation
of the acids formed with O-(4-methoxybenzyl)hydroxylamine) leading to the
O-(4-methoxybenzyl)carbohydroxamic derivatives 47, 89 and 113, respectively.
The target acetohydroxamic acids 2, 9 and 14 were prepared by treating
analogues 47, 89 and 113, respectively with TFA in the presence of Et3SiH in
anhydrous DCM.
For the preparation of the spirocarbocyclic,
N-[(2,6-diketopiperazin-1-yl)glycine hydroxamic acids 19-29, the carboxylic
acids 43, 44, 59, 60, 128, 147 and 148 were converted to the respective
spirocarbocyclic N-[(2,6-dioxopiperazin-1-yl)acetyl]glycine benzyl esters
129-134 (Scheme 11) and 149-153 (Scheme 12), under peptide synthesis
conditions. In detail, the glycine benzyl esters 129-131 and 149-152 were
obtained from the respective carboxylic acids upon reaction with glycine
benzylester (as tosylates), in the presence of ΕDCI.HCl, HOBt, and DIEA in dry
DCM-dry DMF (3:5 v/v). The preparation of glycine benzyl esters 132-134 and 153
was effected by reacting the respective carboxylic acids with N-methylglycine
benzyl ester (as HCl salt), in the presence of CDI and in dry THF. Subsequent
catalytic hydrogenation of the benzyl esters 129-134 and 149-153 led to the
respective N-substituted glycine derivatives 135-140 και 154-158. Treatment of
the latter with O-benzylhydoxylamine hydrochloride, either in the presence of
ΕDCI.HCl, HOBt, and Εt3N or DIEA or CDI and Εt3N, led to the
O-benzylcarbohydroxamic derivatives 141-146 and 159-163, which were converted
to the desired glycine hydroxamic acids 19-29 upon catalytic hydrogenolysis.
In an attempt to decipher why the N-methylation of the carbohydroxamic group of
the acetohydroxamic acids 5 and 6 (2o hydroxamic acids) leads to inactivity
(Table 2), whilst lack of methylation (compounds VIIIa and IX, Table 1) does
not, a series of Ε/Ζ conformational studies on analogues 5, 6, VIIIa and IX was
undertaken, using NMR spetcroscopy and theoretical calculations. The NMR
experiments were conducted at ambient temperature, using DMSO-d6 as solvent.
Based on the NMR results, it can be argued that the 1o hydroxamic acids VIIIa
and IX show a preference for the E-conformation (Ε/Ζ=75/25). Conversely, their
2o N-methylated congeners 5 and 6 adopt exclusively the E-conformation in the
DMSO-d6 solution.
The in-vitro anti-trypanosomal pharmacological results obtained for the
compounds 1-6, 7-18 and 19-29, are as follows:
1. The spirocarbocyclic 2,6-diketopiparazin-1-acetohydroxamic acids 1-4
are very potent against Τ. Brucei, either as free bases or as hydrochlorides
(ΙC50= 32-283 nM, as free bases and ΙC50= 31-253 nM, in the form of
hydrochloride salts).
2. The 2o hydroxamic acids 5 and 6 were found to be inactive against Τ.
Brucei, irrespectively of the form tested (free bases or hydrochloride salts).
Interestingly, these analogues are almost 2000 times less active than their
non-methylated counterparts VIIIa and IX. This dramatic drop in potency can be
probably attributed to the absence of the respective Z-conformer, in the
binding site of the metalloenzyme.
3. The replacement of the spirocarbocyclic ring (adamantano, cyclooctano
and cycloheptano) in compounds VIIIa, Xa and XΙa (Table 1) by benzene
(substituted and unsubstituted) and an alkyl substituent (CH3, (CH2)2CH3,
(CH2)3CH3, compounds 7-11, respectively) lowers, in general, the activity (IC50
= 1,72-19,1 μΜ, as free bases and IC50 = 1,85-12,9 μΜ, as hydrochlorides).
However, a number of these analogues did show good activity (IC50 = 1,72-7,25
μΜ).
4. The alkylation of the basic nitrogen atom of the diketopiperazine
ring of compounds 7-11 by a methyl, n-propyl or n-butyl group (analogues 12-18,
respectively), leads to a substantial improvement of the trypanocidal activity
(ΙC50= 0.47 – 8.16 μΜ). It is noteworthy, that the potency of derivatives 12
and 14-18 is at low micromolar to submicromolar levels (ΙC50= 0.47 – 2.97 μΜ).
5. The introduction of the N-acetylglycinohydroxamic residue
(CH2CON(R)CH2CONHOH, R=H, CH3) on the imidic nitrogen atom of the
spirocarbocyclic 2,6-diketopiperazine scaffolds, leads, in general, to much
less active compounds (Ν-substituted glycinohydroxamic acids, Table 4),
compared to their congeneric acetohydroxamic (CH2CONHOH) bearing residue at the
same position. However, it is noteworthy that the combination of (S)-isobutyl
or (S)-benzyl substitution on the methylenic carbon, located between the aminic
nitrogen atom and the carbonyl of the 2,6-diketopiperazine ring of the
adamantano compound 19, and the concomitant introduction of a methyl group on
the glycinic nitrogen of the side chain, leads to a substantial improvement of
activity. Thus, the derivatives 23 (IC50= 34 nΜ) and 24 (IC50= 53 nΜ), which
are formed by the aforementioned combination of substituents have nanomolar
level potency. Indicatively, analogue 23 having the spiroadamantano
(S)-isobutyl-2,6-diketopiperazine scaffold was 8.3 times more potent than the
respective acetohydroxamic compound 1 (IC50= 283 nM, Table 2).
6. The cytotoxicity of the active spirocarbocyclic
2,6-diketopiperazino-1-acetohydroxamic acids 1-4 (Table 2) and the most active
among the 3-alkyl-3-aryl-2,6-diketopiperazino-1-acetohydroxamic acids 7-18
(Table 3) on mammalian rat L6-cells was found to be almost negligible
(SI=110-2830). Last, low to moderate (SI=16-940) was found to be the
cytotoxicity of the active among the N-substituted glycinohydroxamic acids
19-29.
Keywords:
Trypanosomiasis, T. brucei, Hydroxamic acids, Ε/Ζ conformations, Mammalian rat L6-cells
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
60
Number of pages:
328
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