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Chimeric antigen receptors (CARs) are able to specifically direct T cells to tumor antigens and therapy with anti-CD19 CARs has already cured cancer patients with B-cell lymphomas who have undergone long-term therapy non-successful. Despite this impressive result, the therapy is currently only approved as a last treatment option for blood cancers due to its life-threatening deficiencies. For patient safety and to enable additional application such as the treatment of solid tumors, CAR-T cells must be controllable, e. g. by chemically programmable CARs (cpCARs) regulated by hapten-like compounds.
This thesis reports the synthesis and characterization of such hapten-like compounds. In the first step, seven different warheads with two different spacers were bound to biotin in order to find a suitable warhead for programming the cpCAR.
In a second step, synthetic routes for the three pharmacophores folate, c(RGD), and an RGD peptidomimetic were developed. The routes allow the modification of the pharmacophores with one of the warheads from the first step. CuAAC was chosen as a bioorthogonal approach to link pharmacophores and warheads.
In total, three different pharmacophores were modified with the 1,3-diketone motif of compound 21 leading to 112, 113 and 128. Activation of the T-cell signaling cascade was tested after binding of these hapten-like compounds to the cpCAR in the presence of suitable target structures. For 112, only a slight, non-significant, activation of the T-cell signaling cascade was observed, whereas for 113 and 128, a significant activation of the T-cell signaling cascade was observed.
The poor solubility of the folate compounds led to alternative strategies. Folic acid was exchanged by pteroic acid and the bifunctional, linear compounds were enlarged to trifunctional dendrimers.
Besides the reported regioisomer in 112, a second one, which was not reported to date, occurred by the cyclization of the linear RGD pentapeptide leading to 113.
After the reported synthesis of an RGD peptidomimetic analogous to 128 could not be reproduced, a new synthetic route was developed. It also consists of 17 steps, but reduces the number of linear steps from 13 to 10. Moreover, the developed route contains an asymmetric hydrogenation step and is, compared to the published one, more flexible by the use of the copper-catalyzed azide-alkyne cycloaddition (CuAAC). In addition, an unknown reaction was observed. Instead of the formation of a Schiff base in the reductive amination of 129, an insertion of propargylamine occurred forming 131. The reaction is almost quantitative and in high purity. After requiring no purification, it could be predestined for industrial purposes, such as the synthesis of N-functionalized 1,2-dihydroquinolines or as a building block with various orthogonal functional groups.
Besides the sulfonamide 16, the diketone (21, 27, 31) and lactam compounds (39 – 41), experiments on adapter molecules with further warheads were performed. In the synthesis of a proadapter approach, in which the warhead is formed only after the retro-aldol reaction catalyzed by the mAb, 6 of 10 steps were successfully performed. A newly developed synthesis to keto-sulfonyl and keto-sulfoxide compounds could not be completed but was performed on a small scale to the point of keto-sulfonyl and keto-sulfoxide. Furthermore, a universal synthesis route was designed to allow the introduction of the warhead at the end of the synthesis by acylation. Thus, after 5 shared steps, 3 of them in quantitative yield, different warheads may be introduced. Moreover, this also facilitates the purification and the analysis of the compounds by the absence of tautomerism or labile groups. However, the acylation experiments were not successful with either the acid cyanide or the Weinreb amide.
In summary, this thesis has proven that the 1,3-diketone motif is a suitable warhead for programming the cpCAR, which was developed by Hudecek et al. (unpublished data). The hapten-like compounds 112, 113 and 128 simultaneously bind to integrin ${\alpha}_v{\beta}_3$ and the cpCAR activating the T-cell signaling cascade. The modular synthesis strategy and the use of the bioorthogonal CuAAC allow straightforward access to these valuable immunotherapeutics but revealed the need for an additional purification step to remove copper ions.
While life expectancy increases worldwide, treatment of neurodegenerative diseases such as AD becomes a major task for industrial and academic research. Currently, a treatment of AD is only symptomatical and limited to an early stage of the disease by inhibiting AChE. A cure for AD might even seem far away. A rethinking of other possible targets is therefore necessary. Addressing targets that can influence AD even at later stages might be the key. Even if it is not possible to find a cure for AD, it is of great value for AD patients by providing an effective medication. The suffering of patients and their families might be relieved and remaining years may be spent with less symptoms and restrictions.
It was shown that a combination of hCB2R agonist and BChE inhibitor might exactly be a promising approach to combat AD. In the previous chapters, a first investigation of dual-acting compounds that address both hCB2R and BChE was illustrated (figure 6.1).
A set of over 30 compounds was obtained by applying SARs from BChE inhibitors to a hCB2R
selective agonist developed by AstraZeneca. In a first in vitro evaluation compounds showed
selectivity over hCB1R and AChE. Further investigations could also prove agonism and showed
that unwanted off-target affinity to hMOP receptor could be designed out. The development of
a homology model for hCB2R (based on a novel hCB1R crystal) could further elucidate the
mode of action of the ligand binding. Lastly, first in vivo studies showed a beneficial effect of
selected dual-acting compounds regarding memory and cognition.
Since these first in vivo studies mainly aim for an inhibition of the BChE, it should be the aim
of upcoming projects to proof the relevance of hCB2R agonism in vivo as well. In addition,
pharmacokinetic as well as solubility studies may help to complete the overall picture.
Currently, hybrid-based dual-acting hCB2R agonists and selective BChE inhibitors are under
investigation in our lab. First in vitro evaluations showed improved BChE inhibition and
selectivity over AChE compared to tacrine.78 Future in vitro and in vivo studies will clarify their
usage as drug molecules with regard to hepatotoxicity and blood-brain barrier penetration.
Since the role of hCB2R is not yet completely elucidated, the use of photochromic toolcompounds
becomes an area of interest. These tool-compounds (and their biological effect) can
be triggered upon irradiation with light and thus help to investigate time scales and ligand
binding.
A set of 5-azobenzene benzimidazoles was developed and synthesized. In radioligand binding
studies, affinity towards hCB2R could be increased upon irradiation with UV-light (figure 6.2).
This makes the investigated compounds the first GPCR ligands that can be activated upon
irradiation (not vice versa).
The aim of upcoming research will be the triggering of a certain intrinsic activity by an
“efficacy-switch”. For this purpose, several attempts are currently under investigation: an
introduction of an azobenzene moiety at the 2-position of the benzimidazole core already led to
a slight difference in efficacy upon irradiation with UV light. Another approach going on in our
lab is the development of hCB1R switches based on the selective hCB1R inverse agonist
rimonabant. First in vitro results are not yet available (figure 6.3).
Although the physiological roles of BChE are not yet determined to date, the importance of this enzyme is continuously increasing as it was found to be associated with several disorders like diabetes mellitus type 2, cardiovascular diseases, obesity and especially with Alzheimer’s disease (AD). In consequence, for investigations of BChE’s pathological role in these diseases and to find new medication strategies, the development of selective and potent inhibitors is necessary.
For this purpose, the current work progresses in five chapters on the exploration of the chemical, physical and biochemical properties of tetrahydroquinazoline based carbamates which were previously reported to be selective BChE inhibitors with potency in the low nanomolar range.
1) A Novel Way to Radiolabel Human Butyrylcholinesterase for PET through Irreversible Transfer of the Radiolabeled Moiety:
PET-radiotracers represent an innovative tool to determine the distribution and the expression of a biological target in vivo. BChE lacks to a large degree of such tracers with a few exceptions. In this work, methods were developed to incorporate the radioisotopes 11C and 18F into the carbamate moiety of an tetrahydroquinazoline based inhibitor. In contrast to reversibly acting PET-probes, the described radiotracers were proven by kinetic studies to transfer the radioisotope covalently onto the active site of BChE, thus labeling the enzyme directly and permanently.
2) Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode:
To investigate the role of the tetrahydroquinazoline carrier scaffold on BChE inhibition, carbamate based inhibitors were synthesized. These compounds were successively used to perform kinetic investigations to determine their inhibition mode. Based on these data, a plausible binding model was postulated explaining the influence of the tetrahydroquinazoline carrier scaffold for binding at BChE’s active site just before carbamate transfer takes place. Additionally, these compounds feature neuroprotective properties and prevent oxidative stress induced cell death in their carbamate form as well as after the release of the tetrahydroquinazoline carrier scaffold.
3) Dual Addressing of Butyrylcholinesterase by Targeting the Catalytic Active Site (CAS) and the Peripheral Anionic Site (PAS):
Compounds which are dual-targeting the CAS and the PAS of BChE are the most potent and selective BChE inhibitors to date with inhibition values in the picomolar range. In this work, a strategy is described how to turn tetrahydroquinazoline based carbamates into dual binding BChE inhibitors. These inhibitors feature a carbamate moiety which is covalently transferred onto the CAS of BChE, and in addition provide a second pharmacophore connected via a linker to the carbamate moiety which is proposed to target the PAS. Preliminary results reveal a high tolerance of BChE towards different linker lengths without decrease in affinity.
4) Investigation into Selective Debenzylation and Ring Cleavage of Quinazoline based Heterocycles:
The tetrahydroquinazoline system is well investigated in terms of its synthesis and its selective oxidation. To explore the reactivity of this system, a tetracyclic tetrahydroquinazoline was exposed to common reduction agents. These experiments revealed a high sensitivity of the tetrahydroquinazoline core towards several reduction conditions
5) Experimental and Theoretical Investigation into the Stability of Cyclic Aminals:
Tetrahydroquinazolines are known to degrade in acidic media through hydrolysis of their aminal system; but literature is lacking of a systematic investigation into this behavior. Therefore, different tetrahydroquinazolines were synthesized and exposed to phosphate buffered systems with defined pH-values. A clear increase of the hydrolysis rate of the aminal system was determined in dependency of an increasing acidic media. Computational studies predicted and experimental studies proved that hydrolysis takes place in an acidic environment while the condensation of this system is preferred in neutral or basic aqueous media.
G protein-coupled receptor research looks out for new technologies to elucidate the complex
processes of receptor activation, function and downstream signaling with spatiotemporal
resolution, preferably in living cells and organisms. A thriving approach consists in making use
of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness
and high degree of orthogonality regarding biological processes. This is realized
by the incorporation of molecular photoswitches, which are able to effectively respond to light,
such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic
acetylcholine receptors belong to class A GPCRs and have received special attention in this
regard due to their role as a prototypic pharmacological system and their therapeutic potential.
They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and
thus regulate diverse important biological processes, especially many neurological functions in
our brain.
In this work, the application of photopharmacological tool compounds to muscarinic receptors
is presented, consisting of pharmacophores extended with azobenzene as light-responsive
motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind
concomitantly to the orthosteric and allosteric binding site of the receptor, which is
demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective
photoswitchable ligands, suitable for further ex vivo and in vivo studies.
Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated
extensively with regard to their photochemical behavior and pharmacological profile, outlining
the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho-
fluoro azobenzene. For the first time on a GPCR it was examined, which impact the
different substitution pattern has on both the binding and the activity on the M1 receptor. Results
show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and
F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit
a considerably different biological profile that has to be investigated carefully.
The achievements gained in this study can give important new insights into the binding mode
and time course of activation processes, enabling precise spatial and temporal resolution of the
complex signaling pathway of muscarinic receptors. Due to their role as exemplary model
system, these findings may be useful for the investigation into other therapeutically relevant
GPCRs.
The present thesis concerns the molecular imaging of opioid receptors and human butyrylcholinesterase with the aid of tailored probes, which are suitable for the respective applied imaging techniques. The first part focusses on imaging of opioid receptors with selective probes using total internal reflection- and single molecule fluorescence microscopy. Design and synthesis of the ligands are presented and their pharmacological characterization and application in microscopy experiments are shown. The second part of this thesis focused on the development of 18F-labeled, selective radiotracers for imaging of butyrylcholinesterase via positron emission tomography. The design and synthesis of each a reversible and pseudoirreversible 18F-labeled tracer are presented. After evaluation of the binding properties of each tracer, their initial application in ex vivo autoradiography- and preliminary in vivo microPET studies is described and analyzed.
G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.
Alzheimer’s disease is a complex network of several pathological hallmarks. These characteristics always occur concomitantly and cannot be taken as distinct features of the disease. While there are hypotheses trying to explain the origin and progression of the illness, none of them is able to pinpoint a definitive cause. This fact challenges researchers not to focus on one individual hallmark but, bearing in mind the big picture, target two or more indications at once. This work, therefore, addresses two of the major characteristics of AD: the cholinergic hypothesis and neurotoxic oxidative stress. The former was achieved by targeting the postsynaptic muscarinic M1 acetylcholine receptor to further investigate its pharmacology, and the latter with the synthesis of neuroprotective natural antioxidant hybrids.
The first aim was the design and synthesis of dualsteric agonists of the muscarinic M1 acetylcholine receptor. Activation of this receptor was previously shown to improve AD pathologies like the formation of Aβ and NFTs and protect against oxidative stress and caspase activation. Selectively targeting the M1 receptor is difficult as subtypes M1 – M5 of the muscarinic AChRs largely share the same orthosteric binding pocket. Orthosteric ligands are thus unsuitable for selective activation of one specific subtype. Secondary, allosteric binding sites are more diverse between subtypes. Allosteric ligands are, however, in most cases dependent on an orthosteric ligand to cause downstream signals. Dualsteric ligands thus utilize the characteristics of both orthosteric and allosteric ligands in form of a message-address concept. Bridged by an alkylene-linker, the allosteric part ensures selectivity, whereas the orthosteric moiety initiates receptor activation. Two sets of compounds were synthesised in this sense. In both cases, the orthosteric ligand carbachol is connected to an allosteric ligand via linkers of different chain length. The first set utilizes the selective allosteric M1 agonist TBPB, the second set employs the selective M1 positive allosteric modulator BQCA. Six compounds were obtained in twelve-step syntheses each. For each one, a reference compound lacking the carbachol moiety was synthesised. The dualsteric ligands 1a-c and 2a c were tested in the IP1 assay. The assay revealed that the TBPB-dualsterics 1 are not able to activate the receptor, whereas the respective TBPB-alkyl reference compounds 27 gave signals depending on the length of the alkylene-linker, suggesting allosteric partial agonism of alkyl compounds 27 and no dualsteric binding of the putatively dualsteric compounds 1. The dualsteric BQCA molecules 2, however, activated the receptor as expected. Efficacy of the C5 linked compound 2b was the highest, yet C3 and C8 compounds (2a and 2c) also showed partial agonism. In this case, the reference compounds 31 showed no receptor activation, implying the intended dualsteric binding mode of the BQCA-carbachol compounds 2. Further investigations will be conducted by the working group of Dr. Christian Tränkle at the Department of Pharmacology at the University of Bonn to confirm binding modes and determine affinities as well as selectivity of the synthesised dualsteric compounds.
The second project dealt with the design, synthesis and biological evaluation of neuroprotective esters of the flavonolignan silibinin. While silibinin is already a potent antioxidant, it has been observed that the 7-OH group has a pro-oxidative character, making this position attractive for functionalisation. In order to obtain more potent antioxidants, the pro-oxidative position was esterified with other antioxidant moieties like ferulic acid 35 and derivatives thereof. Seventeen esters of silibinin 32, including pure diastereomers of 7 O feruloylsilibinin (43a and 43b) and a cinnamic acid ester of 2,3-dehydrosilibinin 46, were synthesised by regioselective esterification using acyl chlorides under basic conditions. The physicochemical antioxidant properties were assessed in the FRAP assay. This assay revealed no improvement of the antioxidant properties except for 7-O-dihydrosinapinoylsilibinin 39b. These results, however, do not correlate with the neuroprotective properties determined in the HT-22 hippocampal neuronal cell model. The assay showed overadditive neuroprotective effects of the esters exceeding those of its components and equimolar mixtures with the most potent compounds being 7-O-cinnamoylsilibinin 37a, 7-O-feruloylsilibinin 38a and the acetonide-protected caffeic acid ester 40a. These potent Michael system bearing compounds may be considered as “PAINS”, but the assays used to assess antioxidant and neuroprotective activities were carefully chosen to avoid false positive readouts. The most potent compounds 37a and 38a, as well as the diastereomers 43a and 43b, were further studied in assays related to AD. In vitro ischemia, inhibition of microglial activation, PC12 cell differentiation and inhibition of Aβ42 and τ protein aggregation assays showed similar results in terms of overadditive effects of the synthesised esters. Moreover, the diastereomers 43a and 43b showed differences in their activities against oxytosis (glutamate-induced apoptosis), inhibition of Aβ42 and τ protein aggregation, and PC12 cell differentiation. The stereospecific effect or mode of action against Aβ42 and τ protein aggregation is more pronounced than that of silybin A (32a) and silybin B (32b) reported in literature and needs to be elucidated in future work. Stability measurements in cell culture medium revealed that the esters do not only get hydrolysed but are partially oxidised to their respective 2,3-dehydrosilibinin esters. Because dehydrosilibinin 45 itself is described as a more potent antioxidant than silibinin 32, 7 O cinnamoyl-2,3-dehydrosilibinin 46 was expected to be even more potent than its un-oxidised counterpart 37a in terms of neuroprotection. The oxytosis assay, however, showed that the neurotoxicity of 46 is much more pronounced, especially at higher concentrations, reducing its neuroprotective potential. Dehydrosilibinin esters are therefore inferior to the silibinin esters for application as neuroprotectants, because of the difficulty of their synthesis and their increased neurotoxicity. A synergistic effect of both species (silibinin and the oxidised form) might, however, be possible or even necessary for the pronounced neuroprotective effects of silibinin esters. As the dehydro-species show distinct neuroprotective properties at low concentrations, their continuous formation over time might make an essential contribution to the overall neuroprotection of the synthesised esters. Due to solubility issues for some of the ester compounds, 7-O-cinnamoylsilibinin 37a was converted into a highly soluble hemisuccinate. The vastly improved solubility of 7 O cinnamoyl-23-O-succinylsilibinin 48 was confirmed in shake-flask experiments. Contrary to expectation, stability examinations showed that the succinyl compound 48 is not cleaved to form 7-O-cinnamoylsilibinin 37a. Neuroprotection assays confirmed that 48 is not a prodrug of the corresponding ester. It was determined that the main site of hydrolysis is the 7-position, cleaving 37 to silibinin 32 and cinnamic acid thus reducing the compound’s neuroprotective effects. Nevertheless, the compound still showed neuroprotection at a concentration of 25 µM. The improved solubility might be more beneficial than the higher neuroprotection of the poorly soluble parent compound 37a in vivo. 7 O Cinnamoylsilibinin 37a was further investigated to reduce Aβ25 35 induced learning impairment in mice. While tendencies of improved short-term and long-term memory in the animals were observed, the effects are not yet statistically significant in both Y-maze and passive avoidance tests. A greater number of test subjects is necessary to ensure correctness of the preliminary results presented in this work. However, an effect of ester 37a is observable in vivo, showing blood-brain barrier penetration. The esters synthesised are a novel approach for the treatment of AD as they show strong neuroprotective effects and their hydrolysis products or metabolites are only non-toxic natural products.
Alzheimer’s disease (AD) is the most common form of dementia, and currently, there is no treatment to cure or halt disease progression. Because the one-target strategy focusing on amyloid-β has failed to generate successful pharmaceutical treatment, this work studies natural products with pleiotropic effects focusing on oxidative stress and neuroinflammation as key drivers of disease progression. The central part of this work focused on flavonoids as neuroprotectants. 7-O-Esters of taxifolin and cinnamic or ferulic acid were synthesized and investigated towards their neuroprotective potential addressing aging and disease. 7-O-Feruloyl- and 7-O-cinnamoyltaxifolin showed overadditive effects in oxidative stress-induced assays in the mouse neuronal cell line HT22 and proved to be protective against neuroinflammation in microglial BV-2 cells. The overadditive effect translated to animals using an Aβ25-35-induced memory-impaired AD mouse model where the compounds were able to ameliorate short-term memory defects. While the disease-modifying effects in vivo were observed, the detailed mechanisms of action and intracellular targets of the compounds remained unclear. Hence, a chemical probe of the neuroprotective flavonoid ester 7-O-cinnamoyltaxifolin was developed and applied in an activity-based protein profiling approach. SERCA and ANT-1 were identified as potential targets. Further, chemical modifications on the flavonoids taxifolin, quercetin, and fisetin were performed. The achievements of this work are an important contribution to the use of secondary plant metabolites as neuroprotectants. Chemical modifications increased the neuroprotective effect of the natural products, and distinct intracellular pathways involved in the neuroprotective mechanisms were identified. The results of this work support the use of secondary plant metabolites as potential therapeutics and hint towards new pharmacological targets for the treatment of neurodegenerative disorders.
Alzheimer´s disease (AD) is a neurodegenerative disease and the most common form of dementia with still no preventive or curative treatment. Besides several risk factors, age is one of the major risks for AD and with an aging society, there is an urgent need for disease modifying agents. The strategy to address only one target within the intertwined network of AD failed so far.
Natural products especially the phytochemical flavonoids, which are poly-phenolic natural products, have shown great potential as disease modifying agents against neurodegenerative disorders like Alzheimer´s disease (AD) with activities even in vivo. Flavonoids are produced by many plants and the native Californian plant Eriodictyon californicum is particularly rich in flavonoids. One of the major flavonoids of E. californicum is sterubin, a very potent agent against oxidative stress and inflammation, two hallmarks and drivers of AD and neurodegeneration. Herein, racemic sterubin was synthesized and separated into its pure (R)- and (S)-enantiomer by chiral HPLC. The pure enantiomers showed comparable neuroprotection in vitro with no significant differences. The stereoisomers were configurationally stable in methanol, but fast racemization was observed in culture medium. Moreover, the activity of sterubin was investigated in vivo, in an AD mouse model. Sterubin showed a significant positive impact on short- and long-term memory at low dosages.
A promising concept for the increase of activity of single flavonoids is hybridization with aromatic acids like cinnamic or ferulic acids. Hybridization of the natural products taxifolin and silibinin with cinnamic acid led to an overadditive effect of these compounds in phenotypic screening assays related to neurodegeneration and AD. Because there are more potent agents as taxifolin or silibinin, the hybrids were further developed, and different flavonoid cinnamic acid hybrids were synthesized. The connection between flavonoids and cinnamic acid was achieved by an amide instead of a labile ester to improve the stability towards hydrolysis to gain better “druggability” of the compounds. To investigate the oxidation state of the C-ring of the flavonoid part, the dehydro analogues of the respective hybrids were also synthesized. The compounds show neuroprotection against oxytosis, ferroptosis and ATP-depletion in the murine hippocampal cell line HT22. While no overall trend within the flavanones compared to the flavones could be assigned, the taxifolin and the quercetin derivative were the most active compounds in course of all assays. The quercetin derivate even shows greater activity than the taxifolin derivate in every assay. As desired no hydrolysis product was found in cellular uptake experiments after 4h, whereas different metabolites were found. The last part of this work focused on synthetic bioisoteres of the natural product curcumin. Due to the drawbacks of curcumin and flavonoids arising from poor pharmacokinetics, rapid metabolism and sometimes instability in aqueous medium, we have examined the biological activity of azobenzene compounds designed as bioisoteres of curcumin, carrying the pharmacophoric catechol group of flavonoids. These bioisosteres exceeded their parent compounds in counteracting intracellular oxidative stress, neuroinflammation and amyloid-beta aggregation. By incorporating an azobenzene moiety and the isosteric behaviour to the natural parent compounds, these compounds may act as molecular tools for further investigation towards the molecular mode of action of natural products.
Drug Discovery based on Oxidative Stress and HDAC6 for Treatment of Neurodegenerative Diseases
(2024)
Most antioxidants reported so far only achieved limited success in AD clinical trials. Growing evidences suggest that merely targeting oxidative stress will not be sufficient to fight AD. While multi-target directed ligands could synergistically modulate different steps in the neurodegenerative process, offering a promising potential for treatment of this complex disease.
Fifteen target compounds have been designed by merging melatonin and ferulic acid into the cap group of a tertiary amide HDAC6 inhibitor. Compound 10b was screened as the best hybrid molecule exhibit potent HDAC6 inhibition and potent antioxidant capacity. Compound 10b also alleviated LPS-induced microglia inflammation and led to a switch from neurotoxic M1 to the neuroprotective M2 microglial phenotype. Moreover, compound 10b show pronounced attenuation of spatial working memory and long-term memory damage in an in vivo AD mouse model. Compound 10b can be a potentially effective drug candidate for treatment of AD and its druggability worth to be further studied.
We have designed ten novel neuroprotectants by hybridizing with several common antioxidants, including ferulic acid, melatonin, lipoic acid, and trolox. The trolox hybrid compound exhibited the most potent neuroprotective effects in multiple neuroprotection assays. Besides, we identified the synergistic effects between trolox and vitamin K derivative, and our trolox hybrid compound showed comparable neuroprotection with the mixture of trolox and vitamin K derivative.
We have designed and synthesized 24 quinone derivatives based on five kinds of different quinones including ubiquinone, 2,3,5-trimethyl-1,4-benzoquinone, memoquin, thymoquinone, and anthraquinone. Trimethylbenzoquinone and thymoquinone derivatives showed more potent neuroprotection than other quinones in oxytosis assay. Therefore, trimethylbenzoquinone and thymoquinone derivatives can be used as lead compounds for further mechanism study and drug discovery for treatment of neurodegenerative disease.
We designed a series of photoswitchable HDAC inhibitors, which could be effective molecular tools due to the high spatial and temporal resolution. In total 23 target compounds were synthesized and photophysicochemically characterized. Azoquinoline-based compounds possess more thermally stable cis-isomers in buffer solution, which were further tested in enzyme-based HDAC inhibition assay. However, none of those tested compounds show significant differences in activities between trans-isomers and corresponding cis-isomers.