Learning from the Hantzsch synthesis
|A reaction more than 100 years old continues to teach us how small organic molecules can act as medicines.
Arthur Hantzsch first reported his useful method for pyridine synthesis in Justus Liebigs Annalen der Chemie in 1882 (1). The reaction produces 1,4-dihydropyridines (DHPs), or Hantzsch esters, as isolable intermediates, and he found that these could then be oxidized to pyridines. In the 1970s and 1980s, the DHP nifedipine garnered widespread attention as a drug to treat hypertension. The explosion of interest in this class of molecules was stimulated by their useful application as medicines and the relatively straightforward Hantzsch synthesis permits the preparation of numerous derivatives (2, 3). This allowed for a systematic study of how structure relates to function and also led to the discovery of numerous alternative antihypertensives, as well as several other classes of potential medicines.
I have included this experiment in lectures and laboratory experiments for the past 20 years because it interests students as an example of pharmaceutical technology and summarizes the organic lecture section on enolate chemistry. More recently, the field of study of this class of medicines has allowed me to take the students on their first steps into a wider world: The explanation of how the Hantzsch esters work as medicines requires introducing numerous inter- and multidisciplinary topics. Many of these developments relied on basic chemical principles. In this article, I outline how Hantzsch esters are a vehicle for integrating biochemistry-related topics into an organic chemistry course. Hantzsch esters continue to generate excitement and illustrate how small molecules function as medicines.
The Hantzsch synthesis
The first step, the condensation of acetoacetate with the aryl aldehyde, is known as the Knoevenagel reaction, one of my favorites among organic name reactions. In the second step, the other half of the molecule can be assembled from ammonia and the second equivalent of acetoacetate; the product is an enamine. Such enamines can be isolated, and many are commercially available. The use of enamines was pioneered and popularized by Gilbert Stork, so in my lectures I usually refer to this as the Stork reaction, although to be fair, Stork invented numerous useful organic reactions.
The critical assembly step can be looked at as a conjugate or Michael addition (those names!). Thus, to keep our organic reaction arrows in proper perspective, during the most important step, Knoevenagel is Michaeled by Stork! Cyclization and the final tautomerization have the driving force of a stable ring size and extended cross-conjugation in the final Hantzsch ester. A lot goes on in one reaction flask, and a remarkably large number of variously substituted aldehydes will perform all of these molecular gymnastics in an hour or so in refluxing alcohol. Each step is from sophomore organic chemistry, and the Hantzsch synthesis is a good example to summarize the section on enolate chemistry in the text with a pharmaceutical example that usually appeals to students.
At this writing, four main classes of Hantzsch esters that have useful biological activities have been well studied. The antihypertensives are the most widely studied, with nifedipine (1) being a well-known example (Figure 2). But several other types are being developed.
A variety of products
Hantzsch esters bridge the gap between domains of the proteins that are their molecular targets and offer a template to which a variety of functional groups can be attached. This may explain why the leads for several types of medicines under development were discovered on the basis of Hantzsch esters. The direct synthesis has allowed pharmacologists to determine what molecular components are essential for the esters biological effects. DHPs have emerged as leads for studying multidrug resistance (MDR) in cancer chemotherapy (2) (5), to treat benign prostatic hyperplasia (BPH) (3) (6), and adenosine receptor antagonists (4) (7).
Each of these Hantzsch esters has somewhat different groups attached to a DHP core, as expected, because they all have different molecular targets. The unifying concept is that the Hantzsch synthesis provides a direct entry to molecules that can span the gap in the domain interface of its molecular target. The versatility of the synthesis allows a medicinal chemist to collect a set of analogues relatively quickly to define which groups are most important, and maximize the desired biological activity.
MDR is a problem found in cancer chemotherapy, resulting from the cancer cell pumping the anticancer agent out of itself. A Hantzsch ester (2) has been found that helps overcome MDR. The structural requirements for this biological activity are different from those of the nifedipine antihypertensive agents. Instead of the aryl group in the 4-position of the DHP, an electron-rich double bond provides the greatest activity and is thought to act by preventing the efflux of anti tumor agents.
The halves of the molecule can be assembled one acetoacetate at a time, and then combined, permitting the preparation of numerous asymmetric Hantzsch esters. BPH is a progressive condition characterized by a nodular enlargement of prostatic tissue that obstructs the urethra. BPH has a potential treatment in the Hantzsch ester known as SNAP 5089 (3), which acts selectively with a specific adrenoreceptor.
Adenosine receptors are potential targets for drugs to treat asthma or the cell death that accompanies heart attack and stroke (ischemia). Recently, DHPs (4) have been discovered that have selective A3 adenosine receptor antagonist activity and also act stereoselectively, that is, the 4-S enantiomer is 35 times more effective in binding the A3 adenosine receptor than its mirror image.
The most striking example of stereoselectivity, however, has been observed for analogues of nifedipine. To explain this, though, a little biochemistry has to be integrated into our discussion.
Because of the relative ease of the Hantzsch synthesis, numerous analogues were prepared by several groups soon after the biological activity of nifedipine was established, and the structureactivity relationship (SAR) for antihypertensive activity was developed rather quickly (Figure 3) (2). One well- recognized feature of SAR is that ortho and meta electron- withdrawing groups on the aromatic ring enhance activity, whereas anything at the para position reduces activity. Conformation and absolute configuration are important factors in the biological effects of DHPs. Because most DHPs adopt a boat conformer in the solid state, nautical nomenclature has been widely adopted to discuss the 3-D shape of Hantzsch esters.
For antihypertensive activity, usually the port ester is best with a relatively small alkyl group, but considerable leeway is possible on the starboard side. This results in an asymmetric Hantzsch ester, which can exist in optically active forms. If the molecular target were chiral, it would be expected that pure enantiomers should have different biological effects (8). (You are a chiral reagent, I often tell my students.) To increase our understanding of how the small drug molecule interacts with a molecular target, some discussion of its structure, shape, and function is necessary.
Target: The calcium channel
The calcium channel is a protein, embedded in the cell membrane, that allows for the entry of ions into the cell (3, 9). An introduction to primary and secondary protein structures is found in most commonly used organic chemistry texts, and the calcium channel can be used as an example for this introduction. As molecules go, it is fairly largemolecular weight ~400 kDa. It also has several subunits (9); the one I will focus on binds the Hantzsch ester and is called the a1a subunit. It has four similar parts, commonly called domains or motifs, which are usually designated by Roman numerals. The domains are similar in that their amino acid sequences are predicted to have about the same shape. The shape predicted from the primary sequence is that each motif has six strands that should be a-helices, and which are given Arabic numerals.
We can talk about very precise positions in the a1a subunit. For example, one important part of the protein that binds DHPs is the IIIS6, the sixth strand of the third domain. Among the complicating factors in obtaining crystallographic data are the size of this protein, the fact that it must be bound in a membrane, and that although the motifs are similar, they are not identical. Even though this proteins X-ray structure has not yet been solved, MacKinnon has obtained the X-ray structure of the potassium channel (10), which functions in a similar manner in many respects. There was a surprise here also: Rather than being cylindrical, the protein has an inverted teepee shape. Using the potassium channel structure as a template, Striessnig and co-workers have recently developed a very elegant pictureprobably the best availableof what the calcium channel may look like (Figure 4) (11).
The study of the function of ion channels often involves an experiment called patch clamp analysis, which can measure the current caused by the flow of ions across the cell membrane (12). Using a glass micropipet, it is possible to record this current for a single cell. The 1991 Nobel Prize in Physiology or Medicine was awarded to Erwin Neher and Bert Sakmann for the development of this technique.
The Hantzsch esters also show a fascinating relationship between structure and function: One enantiomer opens the channel, and its mirror image closes it. As shown in Figure 5, the ()-R-enantiomer allows fewer ions to pass through the calcium channel compared with the control, resulting in the lower (less negative) measured electron current. Thus, it is called an antagonist, or calcium channel blocker, and this type of biological action is useful for treating hypertension. Its mirror image, the (+)-S-isomer, increases the current because more ions cross through the calcium channel protein; this molecule is called an agonist.
This is one of the most remarkable examples of stereoselectivity of action that one could expect to stumble across: The mirror images have completely opposite biological effects.
The binding site: Site-directed mutagenesis
Catterall used the technique of changing one amino acid residue at a time in order to define the DHP binding site (13, 14). He recognized that the Hantzsch ester bridged the gap between two domains of the proteins; hence, he called the binding site picture the domain interface model (Figure 6). This may be the clue as to why Hantzsch esters also work as a template for other drug discovery endeavors: Small molecules that bridge a proteins domain interface will significantly affect the way the protein can move, and therefore function. Using a combination of Catteralls essential amino acid binding residues, and the MacKinnon crystal structure, Striessnigs group developed the picture of the probable DHP binding site (Figure 4).
The computational model developed by Schleifer (Figure 7) (15), gives a definite molecular picture of how the medicine may bind the cellular target, especially postulating a specific role for Hantzsch ester binding of tyrosines in the adjacent domains. In my opinion, this represents a new hypothesis generator for the development of new and better drugs based on Hantzsch esters. It is a starting point for arguments about how the Hantzsch esters bind and exert their effect. Very precise experiments can now be designed to test the validity of this model; and as might be expected, this will probably lead to the synthesis of new Hantzsch esters to test the new binding hypotheses.
I dont want to leave the reader with the impression that Hantzsch esters have produced a Panglossian panacea. As do many drugs, antihypertensives of the nifedipine class have recently been reported to have side effects (16, 17), although there is certainly debate about this. The first controversy that emerged was the report of myocardial infarction (heart attack) associated with the use of nifedipine, which is now avoided by development of time-release formulations of the medicine. The second controversy was the report of an incidence of cancer associated with the use of calcium channel blockers in older patients (18, 19); in this case, critics of the study attributed the observations to flaws in the methods used (20). However, these reports should serve as a stimulus for the continuing search for more effective medicines, andrational structure-based design eventually should allow organic and medicinal chemists to produce better Hantzsch esters, with few or no side effects. This is exactly where a better drug receptor picture will allow chemists to guide their imaginations to prepare improved medicines, with a critical tool already in their possession: the Hantzsch synthesis.
The Hantzsch synthesis continues to yield pleasant surprises as we move into a second century of its study. It serves as a vehicle to introduce how a potential medicine is prepared, and how its mechanism of action is studied. This in turn requires the explanation of some of the biochemical techniques used to study a medicines influence: The structure of proteins, their function, and how the drug binds require a discussion of site-directed mutagenesis. Organic chemists need to broaden their horizons to participate in rational structure-based design, and the Hantzsch synthesis and its products provide a good example for students to take their first steps into a wider world.
Nicholas R. Natale is a professor of chemistry at the University of Idaho (Department of Chemistry, 301 Renfrew Hall, POB 442343, Moscow ID 83844-2343; 208-885-6778; nrnatale@ uidaho.edu). His research and teaching interests include synthetic organic methods and medicinal chemistry. His organic methods work focuses on the use of lanthanides and isoxazole lateral metallation;. his medicinal projects include anticancer combilexins, neurotransmitter receptor ligands, and antihypertensive Hantzsch esters. His outreach program, The Chemistry of Winning Teams, uses everyday materials to introduce chemical concepts (www.chem.uidaho.edu/). He completed postdoctoral research at Colorado State University after receiving his B.S. degree and Ph.D., both in chemistry, from Drexel University.