Bifunctional octadentate pseudopeptides as Zirconium-89 chelators for immuno-PET applications.

BACKGROUND: Positron emission tomography (PET) is a highly sensitive method that provides fine resolution images, useful in the field of clinical diagnostics. In this context, Zirconium-89 (89Zr)-based imaging agents have represented a great challenge in molecular imaging with immuno-PET, which employs antibodies (mAbs) as biological vectors. Indeed, immuno-PET requires radionuclides that can be attached to the mAb to provide stable in vivo conjugates, and for this purpose, the radioactive element should have a decay half-life compatible with the time needed for the biodistribution of the immunoglobulin. In this regard, 89Zr is an ideal radioisotope for immuno-PET because its half-life perfectly matches the in vivo pharmacokinetics of mAbs. RESULTS: The main objective of this work was the design and synthesis of a series of bifunctional octadentate pseudopeptides able to generate stable 89Zr complexes. To achieve this, here we investigated hydroxamate, N-methylhydroxamate and catecholate chelating moieties in complexing radioactive zirconium. N-methylhydroxamate proved to be the most effective 89Zr-chelating group. Furthermore, the increased flexibility and hydrophilicity obtained by using polyoxyethylene groups spacing the hydroxamate units led to chelators capable of rapidly forming (15 min) stable and water-soluble complexes with 89Zr under mild reaction conditions (aqueous environment, room temperature, and physiological pH) that are mandatory for complexation reactions involving biomolecules. Additionally, we report challenge experiments with the competitor ligand EDTA and metal ions such as Fe3+, Zn2+ and Cu2+. In all examined conditions, the chelators demonstrated stability against transmetallation. Finally, a maleimide moiety was introduced to apply one of the most promising ligands in bioconjugation reactions through Thiol-Michael chemistry. CONCLUSION: Combining solid phase and solution synthesis techniques, we identified novel 89Zr-chelating molecules with a peptide scaffold. The adopted chemical design allowed modulation of molecular flexibility, hydrophilicity, as well as the decoration with different zirconium chelating groups. Best results in terms of 89Zr-chelating properties were achieved with the N-methyl hydroxamate moiety. The Zirconium complexes obtained with the most effective compounds were water-soluble, stable to transmetallation, and resistant to peptidases for at least 6 days. Further studies are needed to assess the potential of this novel class of molecules as Zirconium-chelating agents for in vivo applications.

Spiropiperidine-Based Oligomycin-Analog Ligands To Counteract the Ischemia-Reperfusion Injury in a Renal Cell Model.

Finding a therapy for ischemia-reperfusion injury, which consists of cell death following restoration of blood flowing into the artery affected by ischemia, is a strong medical need. Nowadays, only the use of broad-spectrum molecular therapies has demonstrated a partial efficacy in protecting the organs following reperfusion, while randomized clinical trials focused on more specific drug targets have failed. In order to overcome this problem, we applied a combination of molecular modeling and chemical synthesis to identify novel spiropiperidine-based structures active in mitochondrial permeability transition pore opening inhibition as a key process to enhance cell survival after blood flow restoration. Our results were confirmed by biological assay on an in vitro cell model on HeLa and human renal proximal tubular epithelial cells and pave the way to further investigation on an in vivo model system.

Novel Aryl Sulfonamide Derivatives as NLRP3 Inflammasome Inhibitors for the Potential Treatment of Cancer.

The NLRP3 inflammasome is a critical component of innate immunity that senses diverse pathogen- and host-derived molecules. However, its aberrant activation has been associated with the pathogenesis of multiple diseases, including cancer. In this study, we designed and synthesized a series of aryl sulfonamide derivatives (ASDs) to inhibit the NLRP3 inflammasome. Among these, compounds 6c, 7n, and 10 specifically inhibited NLRP3 activation at nanomolar concentrations without affecting the activation of the NLRC4 and AIM2 inflammasomes. Furthermore, we demonstrated that these compounds reduce interleukin-1ß (IL-1ß) production in vivo and attenuate melanoma tumor growth. Moreover, metabolic stability in liver microsomes of 6c, 7n, and 10 was studied along with plasma exposure in mice of the most interesting compound 6c. Therefore, we generated potent NLRP3 inflammasome inhibitors, which can be considered in future medicinal chemistry and pharmacological studies aimed at developing a new therapeutic approach for NLRP3 inflammasome-driven cancer.

Non-neuronal TRPA1 encodes mechanical allodynia associated with neurogenic inflammation and partial nerve injury in rats.

BACKGROUND AND PURPOSE: The pro-algesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by a subpopulation of primary sensory neurons, has been implicated in various pain models in mice. However, evidence in rats indicates that TRPA1 conveys nociceptive signals elicited by channel activators, but not those associated with tissue inflammation or nerve injury. Here, in rats, we explored the TRPA1 role in mechanical allodynia associated with stimulation of peptidergic primary sensory neurons (neurogenic inflammation) and moderate (partial sciatic nerve ligation, pSNL) or severe (chronic constriction injury, CCI) sciatic nerve injury. EXPERIMENTAL APPROACH: Acute nociception and mechanical hypersensitivity associated with neurogenic inflammation and sciatic nerve injury (pSNL and CCI) were investigated in rats with TRPA1 pharmacological antagonism or genetic silencing. TRPA1 presence and function were analysed in cultured rat Schwann cells. KEY RESULTS: Hind paw mechanical allodynia (HPMA), but not acute nociception, evoked by local injection of capsaicin or allyl isothiocyanate, the TRP vanilloid 1 (TRPV1) or the TRPA1 activators was mediated by CGRP released from peripheral sensory nerve terminals. CGRP-evoked HPMA was sustained by a ROS-dependent TRPA1 activation, probably in Schwann cells. HPMA evoked by pSNL, but not that evoked by CCI, was mediated by ROS and TRPA1 without the involvement of CGRP. CONCLUSIONS AND IMPLICATIONS: As found in mice, TRPA1 mediates mechanical allodynia associated with neurogenic inflammation and moderate nerve injury in rats. The channel contribution to mechanical hypersensitivity is a common feature in rodents and might be explored in humans.

Identification of small-molecule urea derivatives as PTPC modulators targeting the c subunit of F1/Fo-ATP synthase.

Maintaining a high percentage of living and functional cells in those pathologies in which excessive cell death occurs, such as neurodegenerative disorders and cardiovascular diseases, is one of the most intriguing challenges in the field of biochemical research for drug discovery. Here, mitochondrial permeability transition-driven regulated cell death is the main mechanism of mitochondrial impairment and cell fate; this pathway is still lacking of satisfying pharmacological treatments to counteract its becoming; for this reason, it needs continuous and intense research to find new compounds as modulator of the permeability transition pore complex (PTPC) activity. In this study, we report the identification of small-molecule urea derivatives able to inhibit PTPC opening following calcium overload and selected for future use in cytoprotection.

Novel Mixed NOP/Opioid Receptor Peptide Agonists.

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents.

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Structure-Activity Relationship Studies on Oxazolo[3,4-a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity.

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Biased Agonism at Nociceptin/Orphanin FQ Receptors: A Structure Activity Study on N/OFQ(1-13)-NH2.

Nociceptin/orphanin FQ (N/OFQ) controls different biological functions via selective stimulation of the N/OFQ peptide (NOP) receptor. The pleiotropic actions of N/OFQ may limit the development of NOP ligands as innovative drugs in different therapeutic areas. The pharmacological concept of functional selectivity (aka biased agonism) might be useful for amplifying beneficial actions and/or counteracting side effects. Thus, molecules with large bias factors toward G protein or ß arrestin are required for investigating the translational value of NOP biased modulation. Herein, the biased behavior of a heterogeneous library of NOP-targeting peptide derivatives was evaluated in vitro with the aim to provide possible insights into the structural determinants that govern the selective activation of G protein versus ß-arrestin. Our results demonstrate that lipidation of N/OFQ(1-13)-NH2 is a useful strategy for obtaining G protein biased agonists for the NOP receptor.

Tetrabranched Hetero-Conjugated Peptides as Bifunctional Agonists of the NOP and Mu Opioid Receptors.

The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt1]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.

Synthesis and Biological Activity of Peptide α-Ketoamide Derivatives as Proteasome Inhibitors.

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the ß5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (ß5 IC50 = 7 nM, ß1 IC50 = 60 µM, ß2 IC50 > 100 µM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.

Role of TRPA1 receptors in skin inflammation induced by volatile chemical irritants in mice.

Contact dermatitis is a very common inflammatory reaction in the skin, causing not only aesthetic problems but also loss functionality at work. The molecular mechanisms of contact dermatitis induced by chemical irritants are still unclear. Considering that transient receptor potential channels (TRP) may induce neurogenic inflammation and the exacerbation of inflammatory responses, here we investigated the role of transient receptor potential channel ankyrin type-1 (TRPA1) in skin inflammation evoked by chemical irritants. Ear oedema and nociceptive responses elicited by the topical application of xylene and toluene were measured in Swiss mice, wild type and TRPA1 knockout (Trpa1-/-) C57BL/6 mice. Histological analyses were performed in mice subjected to the ear oedema assay. Topical application of xylene and toluene in the mouse ear induced an edematogenic response (0.113 ±â€¯0.008 mm and 0.067 ±â€¯0.011 mm), compared to vehicle (0.008 ±â€¯0.008 mm), assessed by ear thickness measurements and histological analyses. These responses were prevented by topical pretreatment with a selective TRPA1 antagonist, HC-030031 (% inhibition: xylene 36.8 ±â€¯9.4% and toluene 50.7 ±â€¯11.0%), and by the genetic deletion of TRPA1 ((% inhibition: xylene 66.6 ±â€¯16.7% and toluene 75 ±â€¯0%). In addition, the topical application of xylene and toluene to the mouse paw elicited nociceptive responses, which were significantly reduced by oral treatment with HC-030031 ((% of inhibition: 84.9 ±â€¯1.3% and 27.1 ±â€¯8.0%, respectively); nociceptive responses were almost completely abolished in Trpa1-/-mice. Our data suggest that the activation of TRPA1 could be involved in some of the symptoms of irritant-mediated contact dermatitis, such as oedema, pain and neurogenic inflammation.

The acyl-glucuronide metabolite of ibuprofen has analgesic and anti-inflammatory effects via the TRPA1 channel.

Ibuprofen is a widely used non-steroidal anti-inflammatory drug (NSAID) that exerts analgesic and anti-inflammatory actions. The transient receptor potential ankyrin 1 (TRPA1) channel, expressed primarily in nociceptors, mediates the action of proalgesic and inflammatory agents. Ibuprofen metabolism yields the reactive compound, ibuprofen-acyl glucuronide, which, like other TRPA1 ligands, covalently interacts with macromolecules. To explore whether ibuprofen-acyl glucuronide contributes to the ibuprofen analgesic and anti-inflammatory actions by targeting TRPA1, we used in vitro tools (TRPA1-expressing human and rodent cells) and in vivo mouse models of inflammatory pain. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited calcium responses evoked by reactive TRPA1 agonists, including allyl isothiocyanate (AITC), in cells expressing the recombinant and native human channel and in cultured rat primary sensory neurons. Responses by the non-reactive agonist, menthol, in a mutant human TRPA1 lacking key cysteine-lysine residues, were not affected. In addition, molecular modeling studies evaluating the covalent interaction of ibuprofen-acyl glucuronide with TRPA1 suggested the key cysteine residue C621 as a probable alkylation site for the ligand. Local administration of ibuprofen-acyl glucuronide, but not ibuprofen, in the mouse hind paw attenuated nociception by AITC and other TRPA1 agonists and the early nociceptive response (phase I) to formalin. Systemic ibuprofen-acyl glucuronide and ibuprofen, but not indomethacin, reduced phase I of the formalin response. Carrageenan-evoked allodynia in mice was reduced by local ibuprofen-acyl glucuronide, but not by ibuprofen, whereas both drugs attenuated PGE2 levels. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited the release of IL-8 evoked by AITC from cultured bronchial epithelial cells. The reactive ibuprofen metabolite selectively antagonizes TRPA1, suggesting that this novel action of ibuprofen-acyl glucuronide might contribute to the analgesic and anti-inflammatory activities of the parent drug.

NOP-Targeted Peptide Ligands.

The nociceptin/orphanin FQ (N/OFQ)-N/OFQ peptide (NOP) receptor system is widely distributed at both the peripheral and central level where it modulates important biological functions with increasing therapeutic implications. This chapter wants to provide a comprehensive and updated overview focused on the available structure-activity relationship studies on NOP receptor peptide ligands developed through different rational approaches. Punctual modifications and cyclizations of the N/OFQ sequence have been properly combined furnishing potent NOP selective ligands with different pharmacological activities (full and partial agonists, pure antagonists) and enhanced metabolic stability in vivo. The screening of peptide libraries provided a second family of NOP ligands that have been successfully optimized. Moreover, recent findings suggest the possibility to apply different multimerization strategies for the realization of multi-target NOP/opioid receptor ligands or tetrabranched N/OFQ derivatives with extraordinarily prolonged duration of action in vivo. The diverse approaches led to the identification of important pharmacological tools along with drug candidates currently in clinical development such as Rec 0438 (aka UFP-112) for the treatment of overactive bladder and SER 100 (aka ZP120) for the clinical management of systolic hypertension.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies.

Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label-free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS-evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq-dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.

Design, synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3',4',5'-trimethoxyphenyl)-3-(2'-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach.

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3',4',5'-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC50 values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3',4',5'-trimethoxyphenyl)-2-propen-1-one framework. The structure-activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC50 values of 0.37, 0.16 and 0.17 µM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC50: 18 µM). This derivative also displayed cytotoxic properties (IC50 values ∼1 µM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G2-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3',4',5'-trimethoxyphenyl scaffold.

Discovery of Novel 1,3,8-Triazaspiro[4.5]decane Derivatives That Target the c Subunit of F1/FO-Adenosine Triphosphate (ATP) Synthase for the Treatment of Reperfusion Damage in Myocardial Infarction.

Recent cardiology research studies have reported the role, function, and structure of the mitochondrial permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery, optimization, and structure-activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F1/FO-ATP synthase complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac function upon administration during reperfusion. The selected compounds did not show off-target effects at the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content despite interacting with the ATP synthase complex.

Naphthoquinone amino acid derivatives, synthesis and biological activity as proteasome inhibitors.

The ubiquitin-proteasome system has been largely investigated for its key role in protein degradation mechanisms that regulate both apoptosis and cell division. Because of their antitumour activity, different classes of proteasome inhibitors have been identified to date. Some of these compounds are currently employed in the clinical treatment of several types of cancer among which multiple myeloma. Here, we describe the design, chemistry, biological activity and modelling studies of a large series of amino acid derivatives linked to a naphthoquinone pharmacophoric group through variable spacers. Some analogues showed interesting inhibitory potency for the ß1 and ß5 subunits of the proteasome with IC50 values in the sub-µm range.