The Medicinal Chemist’s Toolbox: An Analysis of Reactions Used in the Pursuit of Drug Candidates

The Medicinal Chemist's Toolbox: An Analysis of Reactions Used in the Pursuit of Drug Candidates

Direct synthesis of Z-unsaturated esters. A useful modification of the horner-emmons olefination.

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain.

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.

Synthetic methods using α-heterosubstituted organometallics

Olefin synthesis with organic phosphonate carbanions

The ability of a series of new cardenolide analogs to produce a positive inotropic effect in isolated, driven, guinea-pig atria was determined. The analogs were all derivatives of digitoxigenin and were prepared by replacing the C17β-lactone ring with a series of open-chain moieties. A range of potency was observed varying from compounds with activities comparable to that of the parent molecule, digitoxigenin, to those which were inactive or which produced a negative inotropic effect. All of the active compounds produced toxic effects at doses close to those which produced maximum stimulant effect. Structure-activity relationships were defined and a model was proposed for that part of the receptor which accommodates the C17 side chain of cardiotonic steroids of the digitalis type. It was hypothesized that the receptor contains an anionic site which lies within a cleft of defied dimensions. High activity was associated with those molecules which poscess a 17β side chain which can fit within the cleft and interact with the anionic site either by a single anion-cation association or by a two-point attachment involving a weak interaction between a partial positive charge on the side chain and the anionic site of the receptor reinforced by the formation of a hydrogen bond. The results were compared with previously published results which described the ability of the analogs to inhibit Na + , K + -activated adenosine triphosphatase. An apparent difference between ability to inhibit the enzyme and ability to induce a positive inotropic effect was observed.

Cardenolide analogs. v. cardiotonic activity of semisynthetic analogs of digitoxigenin

1. In the phenobarbitalized dog, intravenous Benadryl Hydrochloride (1-16 mgm./kgm., at 1-43 mgm./kgm./min.) typically elicited primary vascular depression (median duration, 2 min.) followed by rise in arterial pressure (median duration, > 46 min.). Both phases were basically independent of important arterial reflex zones, vagal cardiac innervation, heart rate changes, peripheral cholinergic mechanisms, and nervous or mechanical respiratory changes. The minimal latent period was ca. 8 sec.

2. The degrees of primary fall were most satisfactorily related, as "probits" fall, with the logarithm of the product of dose per kilogram by its rate of injection. One mgm./kgm. at 0.75 mgm./kgm./min., under the conditions used, would be expected to lower blood pressure by 0.05% to 19% (95% confidence limits). The corresponding dose-rate product lies somewhat above the highest human intraordinary products reviewed, and probably, in corresponding blood level, above ordinary oral doses used.

3. The secondary pressure rise was low-grade (maximum, 26 mm. Hg; median, > 7 mm. Hg), decreased on repetition of injection, and was not clearly correlated with dose. With large doses it was masked by the primary depression for as long as > 1.4hr.

4. Small doses were associated with a variable small cardiac acceleration (maximum, 20%) independent of blood-pressure changes, larger doses with a small primary deceleration (maximum, 22%), masking the acceleration and independent of vagus or carotid nerves. No gross disturbances in cardiac rhythm were observed in limited electro-cardiographic recordings.

5. In the isolated rabbit heart, a dose equal to one of papaverine hydrochloride which elicits slight fluctuations in ventricular contractions and strong durable increase in coronary flow, caused slight to moderate, repeatedly reversible depression of contractions, and a fleeting increase in coronary flow followed by more lasting, mild decrease in flow. The flow changes were not dependent upon mechanical changes and none of the effects was entirely dependent on anti-histamine action.

6. One mgm./kgm. frequently caused slight to moderate, transient stimulation of respiration when injected at rates of 1.2 to 43 mgm./kgm./min. One mgm./kgm. at 19 or more mgm./kgm./min. caused in addition an early restriction in depth of breathing frequently reaching temporary apnea. In such cases, and with larger doses, stimulation was expressed ultimately as acceleration or polypnea, intense and lasting well over an hour with large doses.

Vagotomy eliminated cleanly the restriction in depth and a major part of the acceleration (potentiated pulmonary proprioceptives?). Carotid denervation eliminated a weak chemo- and pressoreceptive stimulation component. In the absence of both mechanisms respiration was essentially undisturbed by large doses.

Cardiovascular and respiratory effects of the antihistamine agent, B-dimethylaminoethyl benzhydryl ether hydrochloride (benadryl hydrochloride).

The ethyl analogue of papaverine (ethaverine) is on the average equal to papaverine in peak potency as a coronary vasodilator in the surviving rabbit heart, when injected quickly. Its action is more durable at suprathreshold dosage so that in total effect summed over time it is more effective.

Relative experimental coronary vasodilator potencies of papaverine and its ethyl analogue, ethaverine (diquinol, perparin)

1. Diacetylcymarol is an acetylated glycoside which is better absorbed than the parent glycoside, cymarol. 2. Diacetyl[19-3H]cymarol was rapidly metabolized and excreted by the rat following intraperitoneal administration. 3. The drug was metabolized extensively to polar compounds with the principal pathway involving loss of the C-19 acetyl group and probable demethylation of the sugar. 4. The bulk of the radioactive material was excreted in the bile and there was little reabsorption. 5. The results show that acetylation was successful in converting the poorly absorbed glycoside, cymarol, into a derivative that was rapidly absorbed from the peritoneal cavity. 6. Following or during absorption, the biologically inactive diacetylcymarol was converted to polar derivatives with potential therapeutic activity. However, subsequent elimination was so rapid that little therapeutic benefit could be expected.

Richard W. Thomas

Papers

Olefin synthesis with organic phosphonate carbanions

Cardenolide analogs. v. cardiotonic activity of semisynthetic analogs of digitoxigenin

Cardiovascular and respiratory effects of the antihistamine agent, B-dimethylaminoethyl benzhydryl ether hydrochloride (benadryl hydrochloride).

Relative experimental coronary vasodilator potencies of papaverine and its ethyl analogue, ethaverine (diquinol, perparin)

Cardenolide analogues. VI. The metabolism of the prodrug diacetylcymarol in the rat