Exploration of the DTrp-NMeLys motif in the search for potent somatostatin antagonists

Previous studies from this laboratory demonstrated that N-methylation at Lys(5) residue in somatostatin octapeptide antagonist analogues increased the GH release inhibition potency by as much as 300%. We have now further investigated N-methylation of this Lys(5) residue in conjunction with a number of N- and C-terminal modifications previously found to give highly potent somatostatin receptor antagonists. Synthetic analogues were tested in a functional assay for their ability to inhibit somatostatin-inhibited GH release from rat pituitary cells in culture and to displace 125I-labeled somatostatin from CHO cells transfected with the five known human somatostatin receptors. Several interesting observations resulted from the study. Replacement of liphophilic Nal(8) at the C-terminus with a hydrophilic His(8) resulted in the increased affinity and selectivity for type 2 receptor to give the most potent antagonist analogue yet discovered (K(i), 1.5 nM), although in the rat pituitary cells inhibitory activity on somatostatin inhibited GH release decreased somewhat. A His(3) substitution within the cyclic portion of the analogues retained pituitary cell potency and affinity for type 2 receptor as did substitution with Bip(8) and Fpa(1). Replacement of Cpa(1) with Iph(1) did not effect the affinity for type 2 receptor significantly, but did decrease the effects on rat cell GH release. Iph(3) within-ring substitution increased the selectivity for sst(2) appreciably although the affinity for that receptor was considerably decreased. Substitution of Npa(3) resulted in good selectivity for sst(2) receptor. Replacement of Nal(8) with D-Trp(8) also increased the selectivity for type 2 receptor. Use of a 'bivalent ligand' approach in which two peptides were joined by 4,4'-biphenyldicarbonyl as a spacer destroyed the affinity for all the subtypes, however, the bivalent ligand formed with the Ahp spacer displayed significant affinity and high selectivity for the type 2 receptor.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic alpha-hydroxymethylamino acids.

New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic alpha-hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to mu and delta opioid receptors. The analogue with (R)-HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to delta and a significant increase of affinity to mu receptors. In contrast, an analogue with (S)-HmPhe in the same position, was very potent and more specific to delta receptors than parent DT I. The analogue with (R)-HmVal at position 5 expressed higher delta affinity and selectivity than parent DT I. The analogue with other possible isomer (S)-HmVal was less selective for delta opioid receptors, as a result of decreasing affinity to delta and increasing affinity to mu receptors. The analogues with (R)- or (S)-HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.     

Structure/activity relationships of C-terminal neuropeptide Y peptide segments and analogues composed of sequence 1-4 linked to 25-36

C-terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single-amino-acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28-36 of neuropeptide Y [neuropeptide Y-(Ac-28-36)-peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y-(Ac-25-36)-peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2-aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28-32 by Ala-Aib-Ala-Aib-Ala decreased receptor binding. However, the corresponding Aib-Ala-Aib-Ala-Aib-substituted analogue and a more flexible analogue with Gly5 at position 28-32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was neuropeptide Y amino acids 1-4 linked to amino acids 25-36 through aminohexanoic acid (Ahx) [neuropeptide Y-(1-4-Ahx-25-36)-peptide].     

5'-Carbamoyl derivatives of 2'-C-methyl-purine nucleosides as selective A1 adenosine receptor agonists: affinity, efficacy, and selectivity for A1 receptor from different species

A series of 5'-carbamoyl and 5'-thionocarbamoyl derivatives of 2'-C-methyl analogues of the A(1) adenosine receptor (A(1)AR) full agonists N(6)-cyclopentyladenosine (CPA), 2-chloro-N(6)-cyclopentyladenosine (CCPA), N(6)-[3-(R)-tetrahydrofuranyl]adenosine (tecadenoson), and 2-chloro analogue (2-Cl-tecadenoson) was synthesized and evaluated for their affinity for adenosine receptor subtypes from bovine, porcine, and human species. In the N(6)-cyclopentylamino series, the 5'-substituted derivatives showed a reduced affinity at the bovine A(1)AR compared to the parent compounds; however, the selectivity for A(1) versus A(2A) receptor was retained or increased. The corresponding N(6)-3-(R)-tetrahydrofuranylamino analogues displayed a very low affinity toward the bovine A(1)AR. The 5'-methylthionocarbamoyl derivative of 2'-Me-CCPA showed the best affinity at porcine A(1)AR with a K(i) value of 13 nM. At human AR subtypes tecadenoson derivatives showed 2.3- to 5-fold lower affinity at A(1)AR and very low affinity at the other subtypes (A(2A), A(2B), and A(3)) compared to the corresponding N(6)-cyclopentyl analogues. The 5'-carbamoyl and 5'-thionocarbamoyl derivatives of 2'-Me-CCPA 3, 4, 7 and tecadenoson derivative 12 were found to be partial A(1) agonists at the porcine receptor. Docking studies explained the lower affinity of N(6)-3-(R)-tetrahydrofuranyl-substituted compounds at bovine A(1)AR compared to that of N(6)-cyclopentyl analogues, showing that the oxygen of the tetrahydrofuranyl ring establishes unfavorable electrostatic interactions with the CO oxygen of Asn254. The low binding affinity of the 2'-C-methyl-N(6)-3-(R)-tetrahydrofuranyl adenosine analogues at human A(1)AR may be ascribed to the presence of unfavorable interactions between the hydrophilic tetrahydrofuranyl ring and the surrounding hydrophobic residues Leu250 (TM6) and Ile274 (TM7).     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

Highly potent and small neuropeptide Y agonist obtained by linking NPY 1-4 via spacer to alpha-helical NPY 25-36

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1-4 linked via epsilon-aminocaproic acid (Aca) to the C-terminal partially alpha-helical peptide amide segment 25-36. NPY 1-4-Aca-25-36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.     

Synthesis of new piperazine-pyridazinone derivatives and their binding affinity toward alpha1-, alpha2-adrenergic and 5-HT1A serotoninergic receptors

We report the design and synthesis of a new class of piperazine-pyridazinone analogues. The arylpiperazine moiety, the length of the spacer, and the terminal molecular fragment were varied to evaluate their influence in determining the affinity of the new compounds toward the alpha1-adrenergic receptor (alpha1-AR), alpha2-adrenergic receptor (alpha2-AR), and the 5-HT1A serotoninergic receptor (5-HT1AR). Biological data showed that most of the compounds have an alpha1-AR affinity in the nanomolar or subnanomolar range, while affinity toward the other two receptors was lower in most cases. However, several of the tested compounds also showed very good (in the nanomolar range) or moderate affinity toward the 5-HT1AR subtype.     

Engineering of insulin receptor isoform-selective insulin analogues

Background

The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/Principal Findings

Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/Significance

We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.     

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4'-phenyl)-phenethyl) analogues of 8-CAC

A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4'-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the mu opioid receptor was achieved though the N-(2-(4'-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to mu and very high binding affinity to kappa opioid receptors was observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.     

Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists

According to a 'bivalent ligand approach' to increase the affinity of the potent argininamide-type NPY Y(1) receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent compound were linked by different spacers via N(G)-acylation at the guanidino groups. A synthetic route for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing nanomolar antagonistic activity and binding affinity to the NPY Y(1) receptor (calcium assay on HEL cells, radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226 derivate showed, surprisingly, no affinity to the NPY Y(1) receptor at all.     

Characterisation of two types of head activator receptor on hydra cells

A head activator (HA) analogue is described which even at high concentrations does not lose its biological activity. By cross-linking two HA molecules over a C8 spacer, the conformation was sufficiently altered, such that self-inactivation of HA by dimerisation was prevented. In addition, the introduction of a tyrosine instead of phenylalanine in one of the two HA molecules allowed radioactive labelling with iodine. This HA bipeptide was used to investigate the effect of HA at different concentrations and as ligand for HA receptor characterisation. We found that low concentrations (0.1-10 pM) sufficed to stimulate interstitial cell mitosis, and that higher concentrations (10-1000 pM) were required for the determination of interstitial cells to nerve cells. Binding of the radioactive HA ligand to living hydra and to purified membrane fractions was saturable and specific. Binding was compatible with HA analogues with a stable monomeric conformation, but less well with dimerising HA and HA analogues. Scatchard and kinetic analyses revealed the presence of at least two types of binding site in the membrane fraction, one with a 'lower' affinity (Kd = 10(-9) M) and one with a 100-fold higher affinity (Kd = 10(-11) M). Autoradiography showed that interstitial cells were differentially labelled, suggesting that the number or types of HA receptors may vary depending on cell cycle status. A mutant of hydra with a multiheaded morphology contained 6-20-times more HA receptors per mg protein than other hydra species or mutants.     

Synthesis and biological activities of phthalocyanine-estradiol conjugates

Phthalocyanine-based photosensitizers, coupled via a 17alpha-ethynyl group to estradiol using Pd(II) as a catalyst, were synthesized and evaluated for their estrogen receptor binding affinity and in vitro photocytotoxicity. The highest receptor binding affinities (RBA=8-13) were observed with lipophilic conjugates coupled via a relative long spacer group while the sulfonated analogues showed little binding affinities (RBA <2). The highest photocytotoxicity was observed with the sulfonated conjugates, the nature of the spacer group did not have a pronounced effect.     

Cardiovascular actions of adenosines, but not adenosine receptors, differ in rat and guinea pig

This study compared the structure-activity relationships of 16 analogues at the A1 and A2 adenosine receptors (A1AR, A2AR) of rat and guinea pig. Radioligand binding studies revealed no marked differences in the affinities of each analogue at the A1AR of brain cortex or the A2AR of brain striatum. Bioassay employing Langendorff heart preparations showed that the guinea pig is more sensitive than the rat to A1AR-mediated slowing of conduction through the atrioventricular node and, in some instances, to A2AR-mediated coronary vasodilation. That difference could reflect factors such as receptor density or efficacy of coupling to effector systems.     

Solid Phase Synthesis of an Analogue of Insulin, A0:R glargine, That Exhibits Decreased Mitogenic Activity

Numerous analogues of insulin have been prepared over the past three decades for use in diabetic therapy. However, only two long-acting insulins have been approved for clinical use. One is Levemir (Novo Nordisk) and the other is Lantus (Sanofi-Aventis). Glargine (commercial name: Lantus) is characterized by a substitution of Gly in place of Asn at the C terminus of the A-chain and addition of two Arg residues to the C terminus of the B-chain. Despite the clinical advantages of glargine, it is not without concern that its increased affinity for the IGF-1 receptor may correlate with increased mitogenic activity. Recently, a systematic study of modified analogues of glargine showed that placement of an extra Arg residue at the N terminus of the A-chain conferred improved insulin:IGF-1 receptor selectivity without significant loss of pharmacological profile. However, as it is difficult to prepare such an analogue in high yield by recombinant DNA methods, we undertook its chemical assembly by our refined solid phase synthesis method. We describe herein its chemical preparation and biological activity in both insulin receptor binding assays and DNA synthesis assays. The synthetic analogue, A0:R glargine, showed slightly reduced affinity for IR-B (twofold) compared to native insulin. In stimulating DNA synthesis, A0:R glargine was slightly less potent compared to insulin or glargine. This result ultimately confirms the previous report that A0:R glargine has a lower potency in mitogenic assays compared to glargine. This glargine analogue thus could be a potential lead compound for drug design and development for the treatment of diabetes.     

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Synthesis of oxytocin antagonists containing conformationally constrained amino acids in position 2

Analogues of oxytocin containing D-Trp, 2-amino-1,2,3,4-tetrahydronaphthalene-1-carboxylic acid (Atc) or 1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid (Car) with R or S configurations in position 2 were synthetized, and their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The peptides were synthetized in the solid phase by using racemates of Car and Atc. The resulting diastereomeric mixtures were separated by means of RP-HPLC. The binding to the oxytocin receptor was somewhat decreased for the Atc isomers and dramatically decreased for both R- and S-Car, while the D-Trp-containing analogue displayed a relatively high receptor affinity. However, the V1 receptor affinities were almost the same as those of the parent peptide for the Car-containing analogues and dramatically decreased for the S-Atc substituted analogue, which has a relatively high OT/V1 receptor selectivity of 44.5.     

Influence of different spacers on the biological profile of a DOTA-somatostatin analogue

Radiolabeled somatostatin analogues have been successfully used for targeted radiotherapy and for imaging of somatostatin receptor (sst1-5)-positive tumors. Nevertheless, these analogues are subject to improving their tumor-to-nontarget ratio to enhance their diagnostic or therapeutic properties, preventing nephrotoxicity. In order to understand the influence of lipophilicity and charge on the pharmacokinetic profile of [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]-somatostatin-based radioligands such as [DOTA,1-Nal3]-octreotide (DOTA-NOC), different spacers (X) based on 8-amino-3,6-dioxaoctanoic acid (PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4), N-acetyl glucosamine (GlcNAc), triglycine, beta-alanine, aspartic acid, and lysine were introduced between the chelator DOTA and the peptide NOC. All DOTA-X-NOC conjugates were synthesized by Fmoc solid-phase synthesis. The partition coefficient (log D) at pH = 7.4 indicated that higher hydrophilicity than [111In-DOTA]-NOC was achieved with the introduction of the mentioned spacers, except with triglycine and beta-alanine. The high affinity of [InIII-DOTA]-NOC for human sst2 (hsst2) was preserved with the structural modifications, while an overall drop for hsst3 affinity was observed, except in the case of [InIII-DOTA]-beta-Ala-NOC. The new conjugates preserved the good affinity for hsst5, except for [InIII-DOTA]-Asn(GlcNAc)-NOC, which showed decreased affinity. A significant 1.2-fold improvement in the specific internalization rate in AR4-2J rat pancreatic tumor cells (sst2 receptor expression) at 4 h was achieved with the introduction of Asp as a spacer in the parent compound. In sst3-expressing HEK cells, the specific internalization rate at 4 h for [111In-DOTA]-NOC (13.1% +/- 0.3%) was maintained with [111In-DOTA]-beta-Ala-NOC (14.0% +/- 1.8%), but the remaining derivatives showed <2% specific internalization. Biodistribution studies were performed with Lewis rats bearing the AR4-2J rat pancreatic tumor. In comparison to [111In-DOTA]-NOC (2.96% +/- 0.48% IA/g), the specific uptake in the tumor at 4 h p.i. was significantly improved for the 111In-labeled sugar analogue (4.17% +/- 0.46% IA/g), which among all the new derivatives presented the best tumor-to-kidney ratio (1.9).