Pierre J. De Clercq

Mechanisms for the selective action of Vitamin D analogs

The non-classical effects of 1,25(OH)(2)D(3) create possible therapeutic applications for immune modulation (e.g. auto-immune diseases and graft rejection), inhibition of cell proliferation (e.g. psoriasis, cancer) and induction of cell differentiation (e.g. cancer). The major drawback related to the use of 1,25(OH)(2)D(3) is its calcemic effect, which prevents the application of pharmacological concentrations. Several analogs are now available that show modest to good selectivity with regard to specific effects (e.g. anticancer or immune effects or bone anabolism versus hypercalcemia) when tested in appropriate in vivo models. The molecular basis for this selectivity is only partially understood and probably a variable mixture of mechanisms.

A Novel Sensitive Colorimetric Assay for Visual Detection of Solid‐Phase Bound Amines

A new simple and efficient method for the detection of incomplete coupling reactions during solid-phase peptide synthesis is decribed. Using p-nitrophenyl ester 1 (NF31), free amino groups can be visually detected on the resin by direct coloring of the beads. A specific feature of the assay resides in the possibility of detection of sterically hindered primary amines.

The Biological Activity of Nonsteroidal Vitamin D Hormone Analogs Lacking Both the C‐ and D‐Rings

1α,25‐dihydroxyvitamin D is a key calcium‐regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A‐ring and side chain, whereas relatively little is known about the requirements of the natural CD‐ring structure for the vitamin D–like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D‐region. This first series of nonsteroidal analogs consists of (1R,3S)‐5‐((Z,2E)‐4‐((1S,3S)‐3‐(4‐hydroxy‐4‐methylpentyl)‐1,2,2,‐trimethylcyclopentyl)‐2‐butenylidene)‐4‐methylenecyclohexane‐1,3‐diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C‐ and D‐rings and by the presence of an unnatural five‐membered ring which we call the E‐ring. KS 176 with the otherwise natural side chain structure of 1α,25(OH)2D3 has between 10 and 30% of the biological activity of 1α,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL‐60 and MG‐63; antiproliferating activity on MCF‐7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD‐rings are not mandatory for the biological activity of 1α,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A‐seco B‐rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.

Biological Activity of CD‐Ring Modified 1α,25‐Dihydroxyvitamin D Analogues: C‐Ring and Five‐Membered D‐Ring Analogues

Nonsteroidal analogues of 1α,25(OH)2D3, lacking either the full five‐membered D ring (C‐ring analogues) or the full six‐membered C ring (D‐ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1α,25(OH)2D3. Maximal superagonistic activity was seen for the C‐ring analogue with a 24(R)‐hydroxyl group in the side chain [30‐ to 60‐fold the activity of 1α,25(OH)2D3]. The 19‐nor‐16‐ene‐26,27‐bishomo C‐ring analogue showed the best ratio of antiproliferative to calcemic effects (1275‐fold better than 1α,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D‐dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR‐ligand complex more resistant against protease digestion than is 1α,25(OH)2D3. In contrast to 20‐epimer steroidal vitamin D analogues, 20‐epimer C‐ring analogues were less potent than analogues with a natural C‐20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1α,25(OH)2D3 despite lower receptor affinity and, for the C‐ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published. (J Bone Miner Res 2000;15;237–252)

Superagonistic Fluorinated Vitamin D3 analogs Stabilize Helix 12 of the Vitamin D receptor

Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.

Interaction of Two Novel 14-Epivitamin D3 Analogs with Vitamin D3 Receptor-Retinoid X Receptor Heterodimers on Vitamin D3 Responsive Elements

This study provides a detailed and exact evaluation of the interactions between vitamin D3 receptor (VDR), retinoid X receptor (RXR), and vitamin D3 responsive elements (VDREs) mediated by two novel 14‐epianalogs of 1,25‐dihydroxyvitamin D [1,25(OH)2D3], 19‐nor‐14‐epi‐23‐yne‐1,25(OH)2D3 (TX 522) and 19‐nor‐14,20‐bisepi‐23‐yne‐1,25(OH)2D3 (TX 527). Both analogs were more potent (14‐ and 75‐fold, respectively) than 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation. However, DNA‐independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA‐dependent assays such as gel shift experiments revealed that those analogs were slightly more potent (3–7 times) than 1,25(OH)2D3 in enhancing binding of VDR‐RXR heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE. The functional consequences of the ligand‐VDR‐RXR‐VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14‐epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)2D3 in COS‐1 and MCF‐7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell‐specific process because maximal transcriptional induction and the half‐maximal transactivation concentration for each reporter construct were different in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF‐7 cell proliferation. These data clearly indicate that superagonistic activity starts beyond the binding of the ligand‐heterodimer (VDR‐RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.

A novel approach to periplanone-B involving an intramolecular Diels-Alder reaction with furan-diene and allene-dienophile

Abstract The 6-step sequence to trienone 2 , a known intermediate for the total synthesis of periplanone-B, involves the conversion of 3 into 4 using dilithiated propadiene, the IMDA of 4 to exo -adducts 5 and 6 , the conversion of 5 to diol 8 via a radical anion promoted c cleavage of the oxygen bridge, and a in-situ low temperature Grob fragmentation to 2 .

Intramolecular diels-alder reaction with furan diene. A new synthesis of 11-keto steroids

Abstract A novel D → BCD → ABCD route to 11-keto steroids is reported involving a high yield stereoselective intramolecular Diels-Alder reaction of furan-diene 5a in water as a key-step. The dienophilic side chain is readily introduced starting from 2 via a sequence involving alkylation with ethyl 4-iodo-3-ethoxycrotonate, reduction and acid hydrolysis. The reduced adduct 8a , obtained in 24 % overall yield from 2-methyl-1,3-cyclocpentanedione, is converted into (±)-adrenosterone ( 16 via base-opening to 9 and further transformation to 13 , the dienediolate equivalent of which is a known intermediate in corticosteroid synthesis.

The Intramolecular Diels-Alder Furan Approach in Synthesis : 11-oxatricyclo|6.2.1.01,6|undec-9-en-5-one

Abstract Although the synthetic potential of the intramolecular variant of the Diels-Alder reaction has now been fully recognized3, little attention has so far been paid to this type of addition using the furan nucleus as the diene partner4–8. The presence of an oxygenated cyclohexene system in several complex natural products (e.g. gibberellic acid, morphine), the diverse possibilities of the substituted 7-oxabicyclo |2.2.1|-2-heptene system for further transformation9, the expected enhanced reactivity and stereoselectivity of the intramolecular cycloaddition and the availability of a wide variety of commercial furan derivatives make the sequence a → b → c particularly attractive in total synthesis.

Intramolecular diels-alder reaction with furan-diene: Total synthesis of (±)-11-ketotestosterone and (±)-adrenosterone

Abstract A novel D → BCD → ABCD route to 11 -keto steroids is reported involving a high yield stereoselective intramolecular Diels-Alder reaction of furan-diene 12 in water as a key-step. The dienophilic side chain is readily introduced starting from 8 via a sequence involving alkylation with ethyl ( E )-3-ethoxy-4-iodo-2-butenoate, reduction and acid hydrolysis. The reduced adduct 14 is further converted into (±)-adrenosterone ( 6 ) via 24 , the dienediolate equivalent of which is a known intermediate in corticosteroid synthesis.