Tetrahydroimidazo[1,2‐a]pyrazine Derivatives: Synthesis and Evaluation as Gαq‐Protein Ligands

Abstract The 5,6,7,8‐tetrahydroimidazo[1,2‐a]pyrazine derivative BIM‐46174 and its dimeric form BIM‐46187 (1) are heterocyclized dipeptides that belong to the very few cell‐permeable compounds known to preferentially silence Gαq proteins. To explore the chemical space of Gαq inhibitors of the BIM chemotype, a combinatorial approach was conducted towards a library of BIM molecules. This library was evaluated in a second messenger‐based fluorescence assay to analyze the activity of Gαq proteins through the determination of intracellular myo‐inositol 1‐phosphate. Structure–activity relationships were deduced and structural requirements for biological activity obtained, which were (i) a redox reactive thiol/disulfane substructure, (ii) an N‐terminal basic amino group, (iii) a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active compounds exhibited cellular toxicity, which was investigated in detail for the prototypical inhibitor 1. This compound affects the structural cytoskeletal dynamics in a Gαq/11‐independent manner.


Introduction
Gp rotein-coupled receptors (GPCRs), also known as seventransmembrane receptors, represent the largest family of cellsurfacer eceptors in eukaryotes. [1] GPCR activation initiates a plethora of intracellular multistep signaling events. [2] About one third of prescription drugso nt he market target GPCRs either as agonistso ra ntagonists. [3] Upon agonist binding, GPCRs undergo conformational changes resulting in the activation of receptor-associated heterotrimeric guanine nucleotidebinding proteins (G proteins), which consist of three different subunits referred to as a, b and g. [4] The capability of acting as molecular switches, thus transducinge xtracellular signals via GPCRsi nto intracellular signal cascades,m akes heterotrimeric Gp roteins vitally important. [5] So far,G PCRsr ather than their associated Gp roteinsh ave been targeted in contemporary drug development. For pathologies,w hicha re characterized by the dysregulation of one specific receptor,t he manipulation of an individual GPCR is suitable. However,i nc ase of complex disorders, such as cancerorpain, which involve multiple receptors and their associated pathways, the interference at the post-receptorl evel appears to be particularly promising and the intracellular Gp roteins can thus be envisaged to serve as potentiald rug targets. [6,7] Furthermore, the direct influence on specific Gp roteins may constitute au seful pharmacological strategy to unravel their role under physiological and pathophysiological conditions. According to the amino acid sequence homology of the Ga subunits,h eterotrimeric Gp roteins are subdivided into four families, Ga s ,G a i/o ,G a q and Ga 12/13 .T he Ga q family comprises four members, that is, Ga q , Ga 11 ,G a 14 and Ga 15/16 ,a mong which the isoformsG a q and Ga 11 are mostc rucial and ubiquitously expressed. [8,9] In their customary role, Ga q familym embers activate their major downstream effector, the enzyme phospholipase C-b (PLCb), leading to hydrolysis of membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol(DAG), whichi nt urn activates protein kinase C, and into myo-inositol 1,4,5-trisphosphate (IP3), which initiates the release of calcium ions from the endoplasmic or sarcoplasmic reticulumi nto the cytosol by opening IP3-sensitive calcium channels. [7,8] The modulation of the Ga q proteins ubfamilyi so fp articular relevance, since its members accountf or aw ide variety of cellular responses, [7,10] leadingt oi mportant physiological functions,s uch as platelet aggregation, [11] insulin-stimulated glu-cose transport, [12] as well as pathophysiological consequences, such as heart failure, [13] and cancer. [6,9,14] Hence, selective and potent modulators are highly required as tool compounds to investigate Gp rotein-mediated signaling or as feasible drug candidates. Prominent modulators of Ga q proteins include, on the one hand, isolatedn atural products, YM-254890 and FR900359, and on the other hand, molecules gained from organic syntheses, the BIM molecules (BIM-46174 and BIM-46187)a nd 27-mer(I860A), all of which represent selective inhibitorsf or Ga q . [7] Amongt hese, the cyclic depsipeptidesY M-254890a nd FR900359, obtained from the fermentation broth of Chromobacterium sp. QS3666 or from the leaves of the plant Ardisia crenata sims,r espectively,exhibit extraordinary selectivity and receive exceptional attention. [6][7][8][15][16] The linear peptide 27-mer(I860A), derived from the phospholipase C-b isoform PLC-b3, has also been identified as as elective Ga q inhibitor, however its molecular mechanism of action still remains unclear. [7,17] The two BIM molecules (Scheme 1) have been previously reported as pan-G protein inhibitors, correspondingt o their ability to likewisei nhibita ll of the four G-protein families. [15,18] This conclusion relied on the compounds' inhibitory interference with two second messenger pathways, the cyclic adenosine-3',5'-monophosphate (cAMP) andt he myo-inositol 1-phosphate (IP1) pathway,a se valuated in human breast cancer MCF7 and melanoma A2058 cell lines, respectively. [15,18] Furthermore, both molecules potently inhibited criticalf unctions in cancer progression, in particularc ell proliferation and cell survival. [18,19] Ad etailed evaluation of BIM action on various GPCR/Gp rotein pairs, co-expressed in different cellular backgrounds such as Chinese hamster ovary (CHO), human embryonic kidney (HEK) 293,a nd CV-1 in origin with SV40 genes (COS) 7c ells revealed preferential silencing of Ga q signaling in ac ellularc ontext-dependent manner. [19] Along the activation pathway,B IM allows GDP release, but prevents GTP entry,t hus trapping Ga q in the nucleotide-free, emptyp ocket conformation. [19] The dimeric BIM-46187 has often been experimentally employed, [20] particularly in order to take advantage of its preferred Ga q inhibition. [21] Despite the apparent usabilityo fB IM-46187, systematic medicinal chemistry approaches are lacking. Just one recent study on BIM fragments demonstrated that distinct structuralr eductions were incompatible with aGa q inhibitory activity. [22] Therefore, we introduceds everalp oints of diversity into the full-size BIM structure and envisaged ac ombinatorial approach towards an extended series of derivatives of BIM-46187 (1). We devised avariety of BIM dimer structuralanalogs, which are ex-pectedt oe xhibit similar redox behavior.H ence, their activity in ac ellular environment should be comparable. Moreover, the thiol function of the BIMm onomer was exchanged for other groups or converted to prodrug forms.T his series of designed compounds, none of which bearing af ree thiol group, was synthesized by means of combinatorial chemistry and subjected to biological investigations on Ga q protein inhibition. We attempted to draw structure-activity relationships, define the BIM pharmacophore and explore the chemicals pace of Ga q inhibitors of the BIM chemotype.

Chemistry
The structure of the BIM monomer can be rationalized as a heterocyclized dipeptide emerged from l-cysteine and l-cyclohexylalanine. The C-terminal carboxyl group is replaced by a4phenyl-substituted imidazole in which the N-1 nitrogen is bridgedt ot he peptidic N atom via an ethylene linker,resulting in a5 ,6,7,8-tetrahydroimidazo[1,2-a]pyrazine core. This scaffold enablesacombinatorial approachb ye ither modifying the heterobicyclic structure or introducing variouss ubstituents at different points of diversity.T he synthesis (Scheme 2) started with the esterification of different d-o rl-configured, N-protected amino acids A with either phenacylb romide or 3-(bromoacetyl)pyridine.T he resulting a-acyloxy ketones B were subjected to aD avidson-type heterocondensation upon treatment with ammonia in refluxingt oluene. 2,4-Disubstituted imidazoles C were reacted with ethyl bromoacetate;t he regioselectivity of this alkylation to occur at N-1 has been recently confirmed by X-ray crystallography. [22] Cbz-protected intermediates D,a fter hydrogenolytic deprotection, underwent an in-stantaneouse ntropy-driven lactamization to E.T he Boc protecting group of other representatives of type D was removed under acidic conditions and ring closure to E occurred at room temperature upon deprotonation of the ammoniumg roup. The further conversion was accomplished by ab orane-promoted reduction,r esulting in relativelys table amine-borane adducts, which could not completely be decomplexed with methanolalone, but in the presenceofp alladium, [23] leading to the desired set of 10 secondary amines F.
Twor epresentatives of this set, that is, the (S)-and( R)-configured cyclohexylalanine derivatives G (Scheme 3) were employed for the synthesis of BIM-46187 (1), [19] its enantiomer 4 and its diastereomers 2 and 3.T he carbodiimide-mediated amide coupling introduced the cysteine unit in H.B oth the tert-butyloxycarbonyl and trityl group were removed contemporaneously under acidic conditions in the presence of excess triisopropylsilane, ah ydride donor, acting as as cavenger of the trityl, and also the tert-butyl cation. [24] The intermediate thiols wereo xidized with iodine, and stereoisomers 1-4 were purifiedb yc olumn chromatography,t hen dissolved in ethyl acetate and precipitateda sh ydrochlorides.
The subseries 5-8 was prepared in order to examine the effect of the basic primary amino group on the bioactivity of the analogs. The NH 2 group was either removed or acetylated, or equippedw ith ab ulky carbamoyl substituent. The first three intermediates I (Scheme 4) were synthesized by means of DCC, while N-acetyl-S-trityl-l-cysteine was introduced with HATU. Prior to oxidation, the detritylation to 5 and 6 and the orthogonal deprotection to 7 and 8 werec arried out as before.
Moreover,p roducts with am odified cystinem ethylene unit were devised whose syntheses are depicted in Scheme 5. The dimericp enicillamine (H-Pen-OH) derivatives 9 and 10 are dimethylated analogs of 1 and 2,r espectively.I nc ompound 10, the structure of the drug d-penicillamine was embedded. We employed homocysteine (H-Hcy-OH) in its double-protected form for the methylene-ethylener eplacement to provide the homocystine derivative 11.I ts absoluteg eometry did not change, but, owing to CIP priority rules, the configurationa t the C-2 carbon is (S).
The starting compound J was also used to prepare intermediates L which were subsequently Boc-deprotectedt ot he final compounds 12-18 (Scheme 6). To obtain 13 from the Boc-Trt-protected precursor,w ea dded triisopropylsilanet of acilitate the liberation of the alcoholicg roup. In the other cases, the same acidic conditions were appliedi nt he final step. The route to the 1,2-diaminopropionate (H-Dap-OH) derivative 14 involved the b-amino protection of Boc-l-Dap-OH, followed by the coupling step andt he simultaneousr elease of both amino groups.T of urnish 15, l-cysteica cid was initially N-Boc-protected, then subjected to coupling conditions in which the free sulfonyl group remained unaffected, and finally deprotected. Products 12-16 can be considered as analogs of the BIM monomer,i nw hich the thiol functioni se xchanged for am ethyl, hydroxy,a mino, sulfo, and methylthiomethyl group. Analogs 17 and 18 are BIM-prodrugs;t he acetamidomethyl (Acm) group of the former might undergo intracellular hydrolytic deacetylation and as ubsequentd ecay to the BIM monomer;t he latter is expected to undergo fragmentation in the reducing environment of the cell to release the corresponding thiol. The synthetic route in Scheme 7f ollowed the aforementioned coupling-deprotection-oxidation sequence. Compounds 19-26 differ with respect to two points of diversity, the amino acid-derivedr esidue R 2 and the aromatic substituent, phenylo r3 -pyridyl, at the imidazole. The structural variability was already inherent in the substitution pattern of 8s econdary amines M.T he achiral precursor for 23 bears two methylg roups at position 8',t he other intermediates M were obtained from (S)-configured amino acids, whereupon (S)serine led to the (R)-configuredf inal product 24.
It was intendedt oe xpand the fused 6-membered ring in 1 to a1 ,4-diazepane substructure in 27 (Scheme 8). Somewhat different reactionc onditions were needed to obtain compound Q,s ince the increased ring strain of the 7-membered ring and the reduced electrophilicity of the ester carbonyl in P, when compared with D (Scheme 2), exacerbated the lactamization. The propylene linker wasf ormed by the borane-promoted reduction of Q.S ubsequent coupling, deprotection and oxidation yielded the envisaged product 27,i nw hich the relative orientation of the phenylg roup and the half-cystinyl residue are slightly shifted.
Analogs with ahigherdegree of conformational heterogeneity werep repareda ss howni nS cheme 9. We either removed the ethylene linker of 1 in the target compound 28 or cut one NÀCb ond in 29.T hese two monocyclic imidazole derivatives possessahigherf lexibility owing to the free rotation about the Ca-C and Ca-N cyclohexylalanine backboneb onds. As in Scheme 8, the synthesis started with compound O which was ethylated at position N-1 to intermediate U.B oth O and U were subjected to hydrogenolytic N-deprotection and the resulting two primary amines V werec onverted via intermediates W to products 28 and 29.

Gp rotein inhibition
We used carbachol (CCh), an agonisto fm uscarinic acetylcholine receptors (mAChRs) to induce Gp rotein signaling in HEK293c ells. The M3 mAChR is expressed in HEK293w ild-type (wt) cells and couples to Ga q and Ga 12 proteinst hereby stimulating phospholipases (PLs) Ca nd Di napertussis toxin-insensitive manner.I np articular, the molecular interaction of Ga q and PLC-b effectuates the cleavage of am embrane phospholipid, that is, PIP2 into DAG and IP3, both acting as important second messengers. IP3 is further hydrolyzed through sequential dephosphorylation to its downstream metabolites myo-inositol 1,4-bisphosphate (IP2) and IP1. The assay which we have employed takes advantages of the inhibition of IP1 degradation by lithium chloride, allowing IP1 to accumulate in the cell, where it can be quantified as as ubstitute for IP3 and am easure of Ga q inhibition. IP1 was determined in acompetitive immunoassay after 2h ours incubation of test compounds (for structures, see Table 1) in ac oncentration of 100 mm followed by 35 min CCh stimulation. The results are showni nF igure 1.
In accordance with previousi nvestigations, [19] the BIM dimer (1)s howedastrong inhibition of the Ga q -dependent IP1 formation and exhibited an IC 50 value of 31.9 mm (see the Supporting Information, Figure S1). The results of the present study revealed, for the first time, the effect of the stereochemistry of isomers on the biological activity.T he (R)-configuration of the cystine substructure was required for Ga q inhibition, but inversion of the cyclohexylalanine configurationw as tolerated. Hence, out of the four stereoisomers, only 1 and 3 (IC 50 = 56.3 mm;s ee the Supporting Information, FigureS1) were potent inhibitors. We assume that 1-4 with highly similar physicochemical properties sharet he cellular uptake capability and specific intracellular drug-protein interactions account for different IP1 accumulation. The positivelyc harged terminal ammonium group was essentialf or Ga q inhibitory activity,s ince neither compounds 5 and 6,t he desamino derivatives of 1 and 3,n or compounds 7 and 8,t he N-protected analogs of 1,w ere active. Subtlec hanges in the cystines ide chain of 1 caused inactivity.T his was the case, when it was equipped with two methyl groups( in 9). In contrast, elongation of the cystine side chain of 1 by one methylene group (in 11)l ed to an unforeseen enhancement of IP1, both in CCh-stimulated cells (Figure 1) and in HEK293c ells without CCh pretreatment( see the Supporting Information, Figure S2). The increased level of cellular inositolp hosphates caused by 11 in aC Ch-independent manner potentially indicates ad irect activation of Ga q or its downstream signaling pathway.
Compound 10,w ith the substructure of the drug d-penicillamine incorporated, was also inactive. IP1 accumulation was maintained after treatment with compounds 12-16.Incontrast to 1 and 3,c ompounds 12-16 are incapable of exhibiting redox reactivity.U nder the assumption that BIM dimers undergo an intracellular reductivec leavage, the thus formed thiol group might be requisite forf urthert ransformationso rd irect Ga q inhibition. Such am ode of action is not possible in case of 12-16.C ompounds 17 and 18 did not serve as successful prodrugs for the BIM monomer.S ince 18 represents ad isulfane, albeit of unsymmetrical structure, we expected ab ioactivation resultingi na ne fficacy comparable to that of 1.H owever,G a q inhibition was not observed.
We next examined whether the desired bioactivity could be achieved in the course of structuralm odifications at the cyclohexylmethyl substructure (in compounds [19][20][21][22][23][24][25].  Figure S1). An increaseo fc onformational flexibility in case of the monocyclic analogs 28 and 29 turned out to be disadvantageous. Taken together,t he following main structuralr equirementsf or active BIM analogs were clarified (i)the redox reactivec ystine/cys- teine substructure, (ii)the N-terminal basic amino group, (iii)the cyclohexylalanine moiety,( iv) ab icyclic skeleton. These considerations providearather limited scope for the future design of related Ga q inhibitors.
The extraordinary potency of YM-254890 and FR900359( IC 50 values of 1.06 mm and 0.77 mm,respectively,see the Supporting Information, Figure S1) was not attained with the BIMa nalogs of this study.F urthermore, their mode of action is also distinct. While BIM-46187 (1)a nd its monomeri nhibitG a q signaling by precluding GTP entry while permitting GDP exit, [19] YM-254890 and FR900359 functiona si nhibitors of GDPd issociation, that is, stabilize Ga q in its inactive GDP-bound state. [5,6] Therefore, YM-254890 and FR900359, but not BIM molecules, specifically disrupt high-affinity agonist binding of Ga q -selective GPCRs, as they impair formation of the nucleotide-free ,empty pocket state', the Gp rotein species required to stabilize this high-affinity interaction.

Cellular toxicity
The toxicityo fa ll compounds was determined by applying a CellTiter-Blue viability assay (Figure 2). It is well-established that Ga q inhibition per se is not linked to cellular toxicity. [6] This finding offered the opportunity to separateefficient Ga q inhibition from adverset oxic effects. On the one hand, an indication for the possible independency of both properties is apparent from the distinct cellular toxicityo fs everalB IM analogs which did not inhibit Ga q (e.g. 10-12, 14, 19, 28 and 29;F igure 1 and Figure 2). On the other hand, significant inhibition was only observed for toxic BIM compounds (1, 3 and 27;F igure 1 and Figure 2). Among the derivatives without af ree N-terminal amino group, some were not toxic (5, 6 and 8,F igure 2). Moreover,r epresentatives bearingp olar groups as part of R 2 (24 and 25)o rt he acidic sulfonate moiety in R 3 (15)d id not affect cell viability either.H ence, az witterionic structure (15,25) might prevent cellular toxicity,a na ssumption to be considered in future attempts towards molecular separation of desired (Ga q inhibition) and unwanted features (cellular toxicity) of BIM analogs.
To investigate whether BIM type inhibitors require Ga q to exert their cell cytotoxic effects, we tooka dvantage of HEK293 cells, depleted by CRISPR/Cas9 of Ga q and Ga 11 subunits of heterotrimeric Gp roteins.C ells treated with the BIM dimer (1) were subjected to non-invasive live cell imaging. We studied the response of wt andG a q/11 KO HEK293 cells to a3 0min long applicationo f1 (30 mm)v ersus DMSO (1:3000) as control. Within 30 min, the cell shape changed, as cells of both cell lines strongly rounded up ( Supporting Information, Figure S3A). From these results we concluded that BIM-induced cell rounding does not requireh eterotrimeric Ga q family proteins. Since the cytoskeleton is an essential cellular component responsible for the maintenance of the morphology and several functions of the cell, we reasoned that cytoskeletal changes might be the origin for the observedt oxicity. To elucidatet he potentialm echanismsu nderlying the compound-induced changes of cell shape, the cytoskeleton was analyzed. For this purpose, we staineda gainstm icrotubules and intermediate filaments with ad irect fluorescent antibodya gainst b-tubulin, and through indirect immunofluorescence against vimentin, respectively.T ov isualize microfilaments, we used rhodamine phalloidin as af luorescent probe for F-actin.T he microtubular network of wt andG a q/11 KO HEK293c ells was not affected by 1 ( Supporting Information, FigureS3B), whereas both wt and Ga q/11 KO cells showed ap referentialp erinuclear accumulation of vimentin andi ntermediate filaments in response to treatment with 1 (Supporting Information, Figure S3C). Them ost prominentc hanges could be observed in phalloidin staining, as both DMSO-treated cell lines displayedp rominent F-actin stress fibers with small protrusions. In clear contrast, compound 1 induced strong roundingu po ft he cells (Supporting Information, FigureS3D) and this was accompanied by predominant cortical actin re-localization, whereas almost neither stress fibers nor actin surroundingt he nucleus of the cells occurred.T hese data demonstrate that the BIM dimer (1)h as prominente ffects on cell shapea nd that these are due to its action on the cytoskeletal components.C ytoskeletal changes in transformed cells are involved in cell proliferating and metastatic capabilities. [25] Noteworthy,o ur data on the Ga q/11 -independenti nfluence of the BIM dimer (1)o nc omponents of the cytoskeleton illustrates an additional mechanism by which BIM molecules act as agentsagainst tumor cells.

Conclusions
These investigationsw ere carriedo ut to exploret he chemical space of Ga q inhibitors with ah eterocyclized dipeptide structure. Although the structure of BIM offers numerouso pportunities for combinatorial modifications and several have been realized in this study,o nly an arrow frame for tolerable structural alterations was recognized. Hence, we defined substructures required for Ga q inhibition and identified two novel bioactive compoundsw hich possess high similarity with the BIM dimer.R edox reactivityw as shown to be requisitef or cellular activity indicating the involvement of intracellularthiols, in particularg lutathione, for bioactivationo fB IM-type disulfanes, probablyc atalyzed by the thioredoxin-glutaredoxin system. [26] Twoc ysteine residues,C ys330 or Cys144, are conserved in all Ga q proteins and might constitute potentialb inding sites for 1,c onsistentw ith the intradomain movement within the targetp rotein. [19] Futures tudies are neededt oa scertain whether ac ovalenti nteraction of Ga q with BIM-typeh omodimers, their reduced monomers or BIM-glutathioneh etero-dimers takes place. Herein we discovered that the prototypical Ga q inhibitor 1 affects the structural cytoskeletal dynamics of treated cells. Moreover,o ur finding provides evidence that BIM-typei nhibitors display their cellular toxicity in aG a q -independentm anner.T he usability of compounds targeting cytoskeletal dynamics has been considered to be limited because they affect av ariety of processes in both cancera nd normal cells. However, there are microtubule-binding compounds that becamev aluable drugs for cancer treatment, in particular vinca domain-binding agents, for example, vincristine, and taxol domain-binding agents, fore xample, paclitaxel. [25] Compound 1 does not exhibit typical molecular features of cytoskeleton-targeting compounds. Accordingly,f uture studies might be focused on structure-activity relationshipsw ith respect to the induction of cytoskeletal changes by BIM-type Ga q inhibitors.

Experimental Section
Detailed descriptions of synthetic procedures and cellular experiments as well as analytical properties of all prepared compounds and biological data are given in the Supporting Information.