Serine-/Cysteine-based sp2-Iminoglycolipids as novel TLR4 agonists: evaluation of their adjuvancy and immunotherapeutic properties in a murine model of asthma

Serine-/Cysteine-Based sp2‑Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma Manuel González-Cuesta, # Alan Chuan-Ying Lai, # Po-Yu Chi, I-Ling Hsu, Nien-Tzu Liu, Ko-Chien Wu, JoséM. García Fernández, Ya-Jen Chang,*and Carmen Ortiz Mellet* Cite This: J. Med. Chem. 2023, 66, 4768−4783 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp2- iminosugar moiety in a prototypic TLR4 agonist, CCL-34, yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8+T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model. ■INTRODUCTION Toll-like receptors (TLRs) are evolutionarily preserved type I transmembrane proteins that recognize conserved pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), representing the first line of host defense. The ability to sense molecular patterns makes TLRs vital regulators for innate immunity, further helping to shape the adaptive immune response by providing key signals to prime naive CD4+T cells toward a specific T helper (Th) profile, like cell-mediated response (Th1) or humoral immune response (Th2). 1−3 Toll-like receptor 4 (TLR4) is one of the most important members of the whole TLR family. It is responsible for the detection of lipopolysaccharides (LPS) from Gram-negative bacteria and plays a key role in the nonspecific inflammatory reaction. The role of TLR4 in driving Th1/Th2 immune response stimulation is complex and multifaceted. TLR4 activation can induce the release of both Th1 (e.g., interferon-γ; IFN-γ) and Th2 cytokines (e.g., interleukines-4 and 13; IL-4 and IL-13) in a context-dependent manner. The precise mechanisms at work are not fully understood, but it is believed to involve the activation of several downstream signaling pathways, including the myeloid differentiation primary response 88 (MyD88)- dependent and TIR-domain-containing adapter-inducing interferon-β(TRIF)-dependent pathways. The resulting Th1/ Th2 polarization is further influenced by various factors, such as the type of TLR4 ligand, the duration and strength of TLR4 activation, and the presence of other co-stimulatory signals. 4,5 Not surprisingly, dysregulated TLR4 signaling contributes to the pathogenesis of many chronic and acute inflammatory diseases, such as asthma, arthritis, cardiovascular disorders, cancer, and sepsis, underlining the importance of TLR4 as a target for therapeutic interventions. 6−8 Both the agonistic and antagonistic aspects of TLR4 signaling pathways are being explored, and a number of clinical trials are currently in progress for a variety of diseases. 9 Efforts in discovering novel TLR4 agonists have mainly been directed toward the development of new vaccine adjuvants, with better properties, improved immune-stimulatory effects, and reduced toxicity, thanks to their ability to trigger a Th1- mediated immune response. 10,11 The most prominent examples are anionic glycolipids related to lipid A, the TLR4 Received: November 28, 2022 Published: March 23, 2023 Articlepubs.acs.org/jmc © 2023 The Authors. Published by American Chemical Society 4768 https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 Downloaded via UNIV DE SEVILLA on May 10, 2023 at 08:26:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. activating motif of bacterial LPS, isolated from either natural or synthetic sources. 12−16 Despite extensive research in the field, monophosphoryl lipid A (MPL; Figure 1), a TLR4 agonist purified from Salmonella minnesota LPS, remains the only adjuvant used in licensed vaccines preventing human papillomavirus (HPV) and hepatitis B virus (HBV) infections. 17−19 MPL is also the adjuvant component of Pollinex Quattro, an allergy vaccine containing the pollen extract which has entered phase II clinical trials for the monotherapy of allergic rhinitis and asthma. 20−23 The underlying concept is that hematopoietic cells (HPCs) and nonhematopoietic stromal or structural airway cells (SACs) express TLR4 in qualitatively differentiated patterns: TLR4 signaling in SACs instructs dendritic cells (DCs) to induce Th2 responses, whereas on HPCs, this receptor can trigger a signaling pathway that programs DCs to polarize the immune response toward Th1 states. Consistent with this view, activation by TLR4 agonists could lead to the attenuation of allergen-triggered Th2 sensitization by enhancing the Th1 response, thus providing protection from airway hyperreactivity (AHR). Although improvement in nasal symptom scores was observed, a relatively high dose (100 μg) was required, and no inhibition of nasal allergen challenge responses compared with placebo was achieved; it was not pursued to phase III. It becomes apparent that exploiting the full potential of TLR4-associated immunity requires small-molecule TLR4 ligands with a specific function such as molecular probes to highlight the specific role of TLR4 in immune system regulation. Serine-based glycolipids, of which CCL-34 (Figure 1) is the archetype, have emerged as a new class of unnatural TLR4 agonists with a strong potential for drug development. 24 Ironically, it was originally conceived as a structural analogue of α-galactosylceramide (α-GalCer or KRN7000; Figure 1), the prototypic ligand of the CD1d receptor in immune system cells. The corresponding CD1d−αGalCer complex binds the T-cell receptor (TCR) on invariant natural killer T cells (iNKT) to induce a strong immune response. CCL-34 comprises a galactopyranose fragment α-linked to a serinescaffolded hydrophobic lipid moiety that contains a linear C12 acyl substituent at the amine group and an N-undecyl amide chain at the carboxylic function of the amino acid component. Compared with other known TLR4 agonists, CCL-34 has the advantages of a defined structure, easier synthesis, and less contamination with bioactive microbial components. It has been shown to modulate differentiation and maturation of myeloid DCs 25 and to elicit anticancer immunity via TLR4. 26,27 Structure−activity relationship studies demonstrated that the sugar portion plays an essential role in the biological properties. The configurational pattern of the monosaccharide admitted substantial variability. Thus, the glucosyl lipid derivative CCL-34-S3 (Figure 1) exhibited a TLR4 agonist potency that was similar to that of CCL-34, suggesting that the stereochemistry of the 4-hydroxyl group is not crucial for activity. However, shifting from α- to βanomeric configuration fully abolished the capability of activating TLR4-dependent signaling. 28 The complications associated with the stereoselective synthesis of α-glycosides, generally requiring tedious and time-consuming procedures, 28−30 and their instability to the action of α-glycosidases represent two important hurdles that may thwart the translation of these glycolipids to the clinic. Figure 1. Structures of monophosphorylated glycolipid A (MPL), α-galactosylceramide (αGarlCer), and the serine-based glycolipids CCL-34 and CCL-34-S3. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4769 We conceived that the abovementioned difficulties could be overcome by replacing the carbohydrate moiety in CCL-34 with an sp2-iminosugar-type surrogate. sp2-Iminosugars are a unique family of glycomimetics characterized by the presence of a pseudoamide-type nitrogen (e.g., a carbamate, urea, thiocarbamate, thiourea, or guanidine nitrogen) in place of the endocyclic oxygen atom in monosaccharides. They emulate not only the structure and function but also the chemistry of monosaccharides, displaying an exacerbated anomeric effect. 31−34 Notably, different from classical iminosugars, sp2- iminosugars can engage in Oand S- glycosylation reactions, affording exclusively the corresponding α-O- or S-glyco- side. 35,36 This property has been previously exploited in the synthesis of specific glycosidase inhibitors and effectors, 37,38 lectin ligands, 39−42 mitogen-activated protein kinase (MAPK) regulators with anti-inflammatory, 43−46 anticancer, 47−49 and antiparasitic activities 50,51 and tumor-associated carbohydrate antigen mimics, 52,53 including a glycoconjugate-based anticancer vaccine. 54 The structural resemblance of the diantennated lipid aglycone in CCL-34 derivatives with the ceramide portion of endogenous glycolipid immunomodulators may enable additional mechanisms contributing to the resolution of the inflammatory process associated with allergy. Thus, glucosylceramide and glycosphingolipids are known to promote or inhibit TLR4-mediated signal transduction in a context-dependent manner by modifying the cell membrane properties. 55,56 Notably, the corresponding α-anomers can further regulate the production of pro-and anti-inflammatory mediators by iNKT cells, 57 a subset of innate-like T cells (CD1d-restricted T cells) that also contribute to allergic inflammation. 58,59 With these considerations in mind, in the present study, we have synthesized serine- and cysteine-based sp2-iminoglycolipids (sp2-IGLs) featuring 5N,6O-oxomethyli- dene-galactonojirimycin (OGJ) and -nojirimycin (ONJ) glycone moieties, namely CCL-34-OGJ (1) and CCL-34- ONJ (2), or the S-glycoside analogues, CCL-34S-OGJ (3) and CCL-34S-ONJ (4), respectively (Figure 2). Assessment of their immunomodulatory properties showed that they represent a novel class of TLR4 agonists with the ability to counteract Th2 sensitization. Investigation of their potential in asthma immunotherapy in a murine model led to the identification of compound 2as a promising candidate for drug development. ■RESULTS AND DISCUSSION Synthesis of sp2-Iminoglycolipids. The preparation of the sp2-IGLs 1−4implies the (thio)glycosylation of the corresponding serine or cysteine lipid with an appropriate OGJ or ONJ pseudoglycosyl donor. The corresponding per-O- acetates 11 and 13 were considered for this purpose. Previous syntheses of the parent sp2-iminosugars, limited to <100 mg batches, proved unsuitable for larger scale production, which seriously hampers access to the target conjugates in sufficient amounts to conduct the relevant in vitro and in vivo studies. Therefore, we first settled on an optimized route for the gramscale preparation of 11 and 13 from commercial D- glucurolactone as a common starting material (Scheme 1). For the synthesis of 11,D-glucuronolactone was first converted into 5-amino-3-O-benzyl-5-deoxy-1,2-isopropyly- dene-α-D-glucofuranose 6through known procedures (5 steps, 22% yield). 60 Subsequent carbonylation with bis- (trichloromethyl)carbonate and diisopropyl ethylamine afforded the bicyclic carbamate 7(80% yield). Acid hydrolysis of the 1,2-O-acetal group in 7with aqueous trifluoroacetic acid proceeded with concomitant furanose−piperidine rearrangement to give the bicyclic piperidine-carbamate compound 8. Isopropylidenation of the 1,2-diol group (→9), followed by trifluoromethanesulfonylation of the 4-hydroxyl group with Figure 2. sp2-IGL structures 1−4(CCL-34-OGJ/ONJ and CCL- 34S-OGJ/ONJ). Scheme 1. Synthesis of the sp2-Iminosugar Glycosyl Donors 11 and 13 a a Reagents and conditions: (a) PPh3and THF−NH4OH, RT, 18 h, quant. (b) BTC, DCM, and DIPEA, RT, 1 h, 80%. (c) TFA−H2O 9:1, RT, 30 min, 75%. (d) PTSA and acetone, 4 Å MS, 50 °C, 4 h, 60%. (e) (i) Tf2O and Py, −30 °C, 1 h; (ii) NaNO2and DCM, RT, 18 h, 50% (two steps). (f) H2, Pd/C, and MeOH, RT, 1 h, quant. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4770 triflic anhydride and reaction of the crude triflate with sodium nitrite in dichloromethane, led to the galacto-configured OGJ derivative 10 in 50% yield. 61,62 Removal of the benzyl and isopropylidene groups by sequential catalytic (Pd/C) hydrogenation and acid hydrolysis, followed by conventional acetylation, provided the OGJ peracetate 11 (5 steps from 6, 18% overall yield, 2 grams of final compound per batch). The gluco-configured glycosyl donor 13, on his side, was prepared in eight steps on a 9 gram scale starting from 5-azido- 5-deoxy-1,2-di-O-isopropylidene-α-D-glucofuranose 5, 63 readily accessible from commercial D-glucurono-6,3-lactone (4 steps, 64% yield). 64 Reduction of the azide and carbonylation of the resulting vicinal aminodiol segment afforded the cyclic carbamate 12. Hydrolysis of the acetonide group with aqueous TFA-promoted conversion gave the corresponding piperidinecarbamate sp2-iminosugar, which was acetylated to give the requested ONJ peracetate 13. 65 (Scheme 1). The preparation of serine-derived lipid acceptors started with commercial N-Fmoc-protected serine (Fmoc−Ser−OH). An amidation reaction with dodecylamine, using DCC and HOBt as coupling reagents, provided the Fmoc-protected compound 14. In the cysteine series, an analogous reaction sequence, starting in this case from the N-Fmoc-S-trityl- protected amino acid (Fmoc−Cys(Trt)−OH), afforded the corresponding dodecylamide 15 and the dianntenated intermediate 16. The final treatment of 16 with TFA and triisopropylsilane (iPr3SiH), as a hydrogen source, 66 yielded the glycosyl acceptor 17 (Scheme 2B) The monoantennated serine lipid precursor 14 was engaged in the glycosidation of 11 and 13 using boron trifluoride etherate (BF3·OEt2) as the promoter. The corresponding O- glycoside derivatives 18 and 19 were thus obtained, with exclusive α-selectivity (1Rconfiguration), in 63−65% yield. Removal of the Fmoc group under mild basic conditions, followed by amidation with lauric chloride and trimethylamine, furnished the CCL-34 mimetics 20 and 21 in a 70−75% yield (two steps). Final deacetylation (NaOMe/MeOH) quantitatively provided CCL-34-OGJ and -ONJ sp2-IGL 1and 2, respectively. The preparation of the cysteine analogues 3and 4was successfully achieved in a two-step sequence involving thioglycosidation of 11 or 13 with the diantennated cysteine lipid 17 in the presence of BF3·OEt2(→22 and 23) followed by Zemplen deacetylation. Attempts to use the analogous diantennated serine lipid as an acceptor proved unpractical due to solubility issues. All the sp2-iminosugar derivatives (final compounds as well as intermediates) showed mass spectrometry and spectroscopic data (1H and 13C NMR) compatible with the proposed structures. Notably, the proton−protoncoupling constants about the six-membered piperidine ring supported that only the α-anomer was present, in agreement with the overwhelming anomeric effect in this family of compounds. The purity of the final compounds 1−4has been confirmed to be >95% by RP−HPLC and combustion microanalysis. In Vitro Cytokine Signature, Adjuvancy Ability, and Signaling Route. Immunological evaluation of the sp2-IGLs 1−4was performed first by assessing their ability to induce the secretion of proinflammatory cytokines in splenocytes isolated from B6 and myeloid differentiation primary response protein (MyD88) KO (MyD88−/−) mice. Since MyD88 is an intermediator of immune response signaling by all TLRs, 67 depletion of inflammatory cytokine level expression on going from wild-type (WT) to MyD88−/−splenocytes can be correlated with TLR involvement. Binding of lipid A to the TLR4/MD-2 complex in immune cells triggers the production of inflammatory cytokines, such as IFNγand IL-6. 68 The serine-based glycolipid CCL-34 likewise promotes an increase in the levels of these two cytokines in splenocytes. 24 We found that the four sp2-IGL-type mimetics synthesized in this work displayed significantly stronger IFNγ expression than the parent compound used as the control in wild-type splenocytes from B6 mice (Figure 3A). The serinebased O-glycolipid mimetics 1and 2further outperformed CCL-34 in enhancing IL-6 expression, whereas the cysteinebased S-glycolipid mimetics 3and 4were less efficient in this case (Figure 3B). We also found that MyD88−/−mice splenocytes treated with the test compounds produced no or very low concentrations of IFNγand IL-6, proving that their Scheme 2. Synthesis of the sp2-IGL 1−4 a a Reagents and conditions: (A) (a) DCC, HOBt, dodecylamine, and THF, RT, 18 h, 90%. (B) (a) DCC, HOBt, dodecylamine, and THF, RT, 18 h, 90%. (b) Piperidine and THF, RT, 10 min. (c) Dodecanoyl chloride, Et3N, and DCM, RT, 18 h, 80% (two steps). (d) TFA−DCM and iPr3SiH, RT, 3 h, 90%. (C) (a) BF3·OEt2,14 or 17, and DCM, RT, 1 h, 63−65%. (b) Piperidine and THF, RT, 10 min. (c) Dodecanoyl chloride, Et3N, and DCM, RT, 18 h, 70−75% (two steps). (d) 1 M NaOMe and MeOH, RT, 1 h, quant. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4771 proinflammatory activity is MyD88-dependent (Figure 3A,B). TLR4-dependent signaling was next confirmed by using human embryonic kidney 293 (HEK-293; 293) cells engineered to express all components of the human TLR4 receptor complex (293/hTLR4A−MD-2−CD14), which is the key for signal transduction by ligands targeting TLR4 (Figure 3C). HEK-293 cells, which do not express any components of the TLR4 receptor complex (TLR4, MD-2, and CD14), were transfected with the genes for human TLR4, MD-2, and CD14. Nontransfected HEK-293 and HEK-293 expressing only the human MD2 and CD14 components (293/hMD-2−CD14) were used as controls. Upon treatment with compounds 1−4, human IL-8 expression was detected in 293/hTLR4A−MD-2− CD14 cells, but not in the control cells, as an unequivocal Figure 3. Immunostimulant function of sp2-IGL is TLR4-dependent but CD1d-independent. (A,B) Splenocytes isolated from B6 (C57BL/6JNarl) and MyD88 KO mice treated with compounds 1−4, with IFNγand IL-6 levels in the culture supernatant measured afterward. (C) HEK-293 (293), 293/hTLR2, 293/hMD2−CD14, and 293/hTLR4A−MD2−CD14 cells treated with compounds 1−4to test for TLR4-dependent agonism, with human IL-8 detected only in the latter as an indicator of TLR4 signaling. (D) Mouse iNKT hybridoma DN3A4−1.2 cells cultured with A20 cells with CD1d expression to assess CD1d-dependent activation, and mouse IL-2 detected as an indicator of CD1d-dependent signaling. (E) DN3A4− 1.2 cells co-cultured with A20−CD1d cells treated with our compounds upon addition to α-GalCer to test for the ability of compounds 1−4to inhibit α-GalCer activity. Data are shown as means ±SEMs of two independent experiments (n= 3 each). *P< 0.05; **P< 0.01. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4772 indicator of TLR4 signaling. To verify the selectivity of TLR4 over TLR2 signaling, the compounds were also tested on HEK-293 cells expressing human TLR2 (293/hTLR2), and no agonist activity (no expression of the human IL-8 reporter molecule) was detected (Figure 3C). We also established that the treatment of compounds 1−4did not cause a significant reduction in cell viability in B6 mouse splenocytes or HEK-293 cells and was well tolerated (MTT assay; Supporting Information Figure S1). Given the structural resemblance of CCL34 and the sp2- IGL-type mimetics 1−4with αGalCer, discerning the offtarget immune activation via the CD1d−TCR pathway is mandatory. By using the mouse iNKT hybridoma DN3A4−1.2 reporter system, we confirmed that none of the test compounds could activate this signaling route (no significant expression of the reporter interleukine IL-2 was detected; Figure 3D). Further, neither treatment with CCL34 nor with any of the analogues 1−4reduced IL-2 expression levels after α-GalCer stimulation of the mouse iNKT hybridoma, supporting that they do not compete with α-GalCer for CD1d binding (Figure 3E). Figure 4. Compound 2-promoted BMDC maturation is dose-dependent and MyD88-dependent. (A,B) BMDCs from B6 (WT) and MyD88 KO mice were treated with compound 2, and the expression of CD86 was assessed using flow cytometry. (C) Expression of IL-12p40 in the culture supernatants of WT and MyD88 KO BMDCs was assessed. Data are shown as means ±SEMs of two independent experiments (n= 4 each). *P< 0.05. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4773 The serine-based glycolipid mimetic 2, exhibiting an immunostimulatory activity in vitro higher than the parent compound CCL34, was next assessed for its ability to induce the maturation of bone marrow-derived dendritic cells (BMDCs). Our results showed that compound 2displayed a dose-dependent effect in inducing the expression of the cluster of differentiation 86 (CD86), a key mediator of T-cell activation and survival (Figure 4A) in WT mouse BMDCs. 69 A parallel assay using BMDCs isolated from MyD88 KO mice evidenced much lower levels of CD86 upon treatment with 50 μM of 2, consistent with a mechanism of action implying TLR4 (Figure 4B). Similarly, the levels of IL-12p40 secretion, a chemoattractant for macrophages, in the WT BMDC culture supernatant increased in the presence of compound 2in a dose-dependent manner, which was not the case for MyD88−/−BMDCs (Figure 4C). The adjuvancy function of CCL34 and compounds 1−4was additionally evaluated in human peripheral blood mononuclear cells (PBMCs) from two different donors, A and B, via the detection of IFNγand IL-6. Compounds 1and 2induced expression levels of both IFNγ(Figure 5A,C) and IL-6 (Figure 5B,D) that rivaled the performance of CCL34, supporting a T- helper 1 (Th1)-biased trait. The cysteine-based derivatives 3 and 4exhibited lower immunostimulatory potency in this assay. In Vivo Evaluation of the Immunostimulatory Activity. The in vivo potential of the sp2-IGLs 1−4as adjuvants was initially judged by determining their capacity to induce the production of IFNγand IL-4 as archetypical mediators of humoral and immune responses, respectively, in WT mice. 70 The levels of both cytokines in serum were significantly increased (Figure 6A,B) at 2 h post-treatment (20 μg compound/g mouse weight, intraperitoneal (i.p.) administration) and remained high after 18 h. Compound 2was found to be the most efficient in the series at increasing the mRNA level of Cd86 in the spleen, signifying its capability to promote DC maturation (Figure 6C). All compounds could also promote a rise in the mRNA level of Il-4, with compound 3showing the strongest effect in this case (Figure 6D). To demonstrate the adjuvant properties of the serine-based sp2-IGLs, we tested their ability to promote ovalbumin (OVA)-specific proliferation of CD8+T cells in the OT-1 adoptive transfer mouse model. 71 Briefly, splenocytes from this mouse model express an OVA-specific T cell receptor. Upon OVA binding, CD8+T cell proliferation is induced, a characteristic feature of an adaptive immune response. We found that mixing OVA with either compound 1or 2 significantly enhanced OVA-specific CD8+T cell proliferation (Supporting Information Figures S2 and 6E) by 10- and 5-fold, respectively, supporting a strong immunostimulatory activity (Figure 6F). In Vivo Evaluation of the Immunotherapeutic Potential against Asthma. The ability of compounds 1−4 to induce high expression levels of IFNγin vivo through the TLR4 pathway, meaning a Th1-biased profile, suggests their potential to counteract Th2-dominated inflammatory pathologies, such as asthma. 72 We have explored this notion in the OVA-induced AHR mouse model. 73 After OVA sensitization, compounds 1−4(20 μg/g mouse weight, i.p.) were used to treat mice. With the administration of increasing concentrations of methacoline, these mice developed bronchoconstriction and consequently enhanced pulmonary resistance (RL). Interestingly, RLvalues were curtailed by as much as 50% upon treatment with compounds 1−4(Figure 7A). Additionally, determination of the levels of the Th2 cytokines IL-4 and IL-13, which are considered instigators of AHR and airway inflammation, in the bronchoalveolar fluid (BALF) and in the lungs was additionally conducted. We found that all Figure 5. Compounds 1−4induce strong IFNγand IL6 expression in PMBCs from different donors. PBMCs from two donors were treated with compounds 1−4. IFNγin the culture supernatants of PBMCs from donors A (A) and B (C), and IL-6 in the culture supernatants from donors A (B) and B (D) were assessed. Data are shown as means ±SEMs of two independent experiments (n= 2−5 each). *P< 0.05; **P< 0.01. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4774 compounds reduced the expression levels of IL-4 in BALF, although data only reached significance for compounds 2−4 (Figure 7B), and a similar trend was observed in the lungs. In the case of IL-13, only the serine-based derivatives 1and 2 were efficient at decreasing expression levels in both BALF and lungs, which agrees with their stronger Th1-biased immunomodulatory character (Figure 7C). ■CONCLUSIONS The results above-discussed show that (thio)glycosidation of sp2-iminosugar glycosyl donors with serine- or cysteine-based lipid acceptors provides an efficient entry to bioactive glycolipid mimetics, namely sp2-IGLs. In these compounds, the sugar portion is replaced by a piperidine−carbamate bicyclic moiety, whose hydroxylation and configurational profiles can be predetermined. Notably, the glycosylation reaction proceeds with total α-stereoselectivity, which was here exploited to synthesize sp2-IGL analogues of the serine-based glycolipid CCL-34, a non-LPS-related TLR4 agonist. Evaluation of the immunomodulatory properties of the new sp2-IGLs supports their TLR4 agonistic character both in vitro and in vivo, with a cytokine signature characterized by high expression levels of IFNγ, suggesting a Th1-biased profile. Interestingly, the compounds were found to counteract the Th2 proinflammatory context in a mouse model of OVA-induced respiratory hyperreactivity mimicking asthma, promoting crisis resolution. They possess well-defined chemical structures, are chemically stable, are expected to be metabolically inert, and can be prepared through efficient and scalable experimental procedures suitable for optimization strategies. Investigation of this novel family of immunomodulators in other immune- Figure 6. Compounds 1−4possess strong adjuvancy function and promote DC and B cell maturation in WT mice and OVA-specific CD8+T cell proliferation in the OT-1 adoptive transfer mouse model. (A−D) B6 mice treated with compounds 1−4(20 μg compound/g mouse weight, i.p. administration). Sera were collected at 2 and 18 h post-treatment of compounds, and IFNγ(A) and IL-4 (B) protein concentrations were measured. Spleens from treated mice were also collected, and the mRNA levels of Cd86 (C) and Il4 (D), normalized to the glyceraldehyde-3- phosphate dehydrogenase (Gaphd) level, were analyzed. (E,F) Splenocytes isolated from OT-1 mice, in which OVA-specific CD8+T cells could be expanded by the antigen OVA, labeled with CFSE dye, and adoptively transferred into B6 mice. Recipient B6 mice were then treated with OVA257−264 peptide mixed with compound 1or 2, and the proliferation of OVA-specific CD8+T cells was analyzed using flow cytometry. (E) Experimental scheme. (F) Percentage of proliferating OVA-specific CD8+T cells. Data are shown as means ±SEMs of two independent experiments (n= 3−8 each). *P< 0.05; **P< 0.01; ***P< 0.001. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4775 compromised pathologies, such as infectious diseases and cancer, is currently sought in our laboratories. ■EXPERIMENTAL SECTION Chemistry. All chemicals were of reagent grade and used without further purification unless otherwise specified. The experimental data for 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-glucofuranose (5), 60 5- amino-5-deoxy-1,2-O-isopropylidene-α-D-glucofuranose 5,6-(cyclic carbamate) (12), 65 (1R)-1,2,3,4-tetra-O-acetyl-5N,6O-oxomethylide- negalactonojirimycin (11), 74 and (1R)-1,2,3,4-tetra-O-acetyl-5N,6O- oxomethylidenenojirimycin (13) 65 were identical to those previously reported. CCL34 was prepared as previously described. 28 Optical rotations were measured at 20 ±2°C in 1 dm tubes on a Jasco P- 2000 polarimeter using a sodium lamp (λ589 nm). IR spectra were recorded on a JASCO FTIR-410 device. UV spectra were recorded on a JASCO V-630 instrument; the unit for εvalues: mM−1cm−1.1H (and 13C NMR) spectra were recorded at 500 (125.7) MHz with a Bruker 500 DRX magnet. 1D TOCSY, 2D COSY, HMQC, and HSQC experiments were used to assist with NMR assignments. Thinlayer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F254 Merck with visualization by UV light and by charring with ethanolic 10% H2SO4and 0.1% ninhydrin. Column chromatography was carried out on Silice 60 A.C.C. Chromagel (SDS 70−200 and 35−70 μm) for preparative purposes. Mass spectra were carried out on a Bruker Daltonics Esquire6000. The samples were introduced via a solid probe heated from 30 to 280 °C. ESI was used as the ionization source (electrospray ionization), and methanol was used as the solvent. The samples were introduced via direct injection using a Cole-Parmer syringe at a flow rate of 2 μL/min. Ions were scanned between 300 and 3000 Da with a scan speed of 13,000 Da/s at unit resolution using resonance ejection at the multipole resonance of one-third of the radio frequency (Ω= 781.25 kHz). Highresolution mass spectrometer (HRMS) spectra were recorded on a Bruker MaXis Impact or Agilent Technologies 6210 time-of-flight LC/MS spectrometers using positive or negative ESI. Elemental analyses were carried out at the Instituto de Investigaciones Quimicas (Sevilla, Spain) using an elemental analyser, the Leco CHNS-932. The analytical results for C, H, N, and S were within ±0.4 of the theoretical values. Nα-Lauryl-O-(1R-5N,6O-oxomethylidenegalactonojirimycinyl)- N-dodecyl-L-serinamide (1). Compound 1was obtained by the treatment of a solution of 20 (45 mg, 0.058 mmol) in MeOH (1 mL) with methanolic 1 M NaOMe (42 μL), followed by neutralization with the Amberlite IR120 H+cation-exchange resin, filtration, and evaporation. Yield: 33 mg (quant). Rf0.60 (70:10:1 DCM−MeOH− H2O). 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Synthesis and Comparative Glycosidase Inhibitory Properties of Reducing Castanospermine Analogues. Eur. J. Org. Chem. 2005,2005, 2903−2913. Journal of Medicinal Chemistry pubs.acs.org/jmc Article https://doi.org/10.1021/acs.jmedchem.2c01948 J. Med. Chem. 2023, 66, 4768−4783 4783 Recommended by ACS Self-Adjuvanting Protein Vaccine Conjugated with a Novel Synthetic TLR4 Agonist on Virus-Like Liposome Induces Potent Immunity against SARS-CoV-2 Dong Ding, Jun Guo, et al. JANUARY 10, 2023 JOURNAL OF MEDICINAL CHEMISTRY READ Configuration-Specific Antibody for Bacterial Heptosylation: An Antiadhesion Therapeutic Strategy Xiang Li, Hong-Gang Hu, et al. DECEMBER 21, 2022 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY READ Structure–Activity Relationship Study of Momordica Saponin II Derivatives as Vaccine Adjuvants Hyunjung Kim, Pengfei Wang, et al. 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