RESEARCH
New cyclic AMP analogs show high activity Some analogs trigger hormone release more specifically than parent compound, raising possibility of control New analogs of cyclic AMP may lead the way to control of diseases related to disruption of the body's hormone system. Some of the analogs are not only more active than c-AMP ( adenosine-S^S'-monophosphate ), but they are also more specific in their action. Results of preliminary studies of activities of the new compounds were disclosed at a New York Academy of Sciences meeting in New York City (C&EN, Nov. 16, page 37) by physician Georges Cehovic, Collège de France, Paris, who confirmed the biological activities of the new molecules synthesized by chemist Theodore Posternak at Université de Genève (Switzerland). Their recent work is published in a November issue of Comptes Rendus Hebdomadaires des Seances de l'Académie des Sciences, Série D. Other scientists at the New York meeting shed further light on how cAMP works in biological systems. And from University of Washington, Seattle, comes evidence on the steps by which c-AMP links hormones with protein synthesis (see page 3 8 ) . Although Dr. Earl W. Sutherland discovered the ubiquitous regulator of body chemistry 14 years ago, research on c-AMP's method and diversity of action didn't accelerate until the mid196(ys. The chemical is found in bacteria and other microorganisms, and in all animal tissue-but not at all in plants. The nucleotide plays a role in both hormone release and mode of actionbut not necessarily both roles with the same hormone. Some hormones are released, and possibly caused to be synthesized, by c-AMP. When a hormone arrives at its target tissue, the hormone activates the enzyme adenyl cyclase, which is bound to the cell's plasma membrane. Adenyl cyclase catalyzes the synthesis of c-AMP inside the cell, and the nucleotide then causes the specific effect attributed to the hormone. C-AMP is thus the socalled second messenger of the hormone. Besides developing the second-messenger concept, Dr. Sutherland and his colleagues also found that the enzyme phosphodiesterase is responsible for c-AMP's degradation. The balance between adenyl cyclase and phosphodiesterase may be responsible for
regulating c-AMP levels in the body and in the cells. Diverse functions. Cyclic AMP has been implicated in muscle function, gastric and enzyme secretion, central nervous system function, and in cellular metabolism. Many scientists believe that if the action of c-AMP can be controlled, the body's responses to many hormones can thereby be controlled. Although c-AMP has diverse functions, some of the nucleotide's analogs synthesized by Dr. Posternak may act more specifically. Development of analogs that act in specific organs or that stimulate specific hormones may thus be a key to controlling many life processes. The in vitro activity studies of Dr. Cehovic have been carried out with rat anterior pituitaries and the release of thyrotrophin-stimulating hormone (TSH), growth hormone ( G H ) , and prolactin (PR). Most of the new derivatives are more active than c-AMP on both GH and TSH release. The compounds Dr. Posternak prepared for Dr. Cehovic's activity work included derivatives of c-AMP with monomethyl, dimethyl, η-butyl, and tert-butyl substitutions on the amino nitrogen at carbon-6 of the adenine group. Other derivatives included isoc-AMP (ribose at N-3 of adenine), 2amino-c-AMP, and thio, thiomethyl, hydroxy, and amino derivatives at carbon-8. Dr. Posternak synthesized the C-6 amino derivatives from 6-chloro9 - ( ribo-β -D-furanosyl-2 / ,3 / -isopropylidene )-purine by reactions with
mono- or dialkylamines. The adeno sine derivative obtained is phosphorylated at the 5' position. After remov ing the isopropylidene group, the sub stituted AMP is cyclized to a 3',5'phosphodiester by methods that Dr. Posternak devised. Some butyryl derivatives of the ana logs were also prepared. For some of the butyryl compounds, a better pene tration into the cell has been postulated by the scientists. In particular, 6-N2'-0-dibutyryl-8-thio-c-AMP shows interesting biological properties, Dr. Cehovic points out. The derivative presented the highest GH release activ ity of all the derivatives tested. Activities. Iso-c-AMP is very ac tive on the release of TSH but far less active on GH. "Also, iso-c-AMP shows a slightly decreased activity in PR release compared to the basic nu cleotide. Derivatives that are much more active than c-AMP on GH and TSH release show no important modi fications on PR release," he notes. The absence of any significant ac tion of c-AMP and its derivatives on PR release can be related to the par ticular conditions of this hormone's release mechanism, Dr. Cehovic ex plains. "We have observed that cy clic guanosine-S'jS'-monophosphate (cGMP, the only other cyclic nucleotide known to occur naturally) and cyclic inosine-S'^'-monophosphate produce a significant release and synthesis of PR in female rats," he adds. The derivative 6-N-2'-0-dibutyrylc-AMP (DBC) shows variable activity at increased concentrations. The vari-
NOV. 30, 1970 C&EN 29
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ability can be related to the recent observation of spontaneous partial decomposition of DBC in aqueous buffers to monobutyryl-c-AMP, butyrate, and c-AMP, the scientists think. In fact, this decomposition of DBC spurred the scientists to synthesize the other derivatives, which are more stable compounds. Except for the terf-butyl analog, the derivatives with alkyl groups on the amino nitrogen at C-6 give activities on both GH and TSH release superior to that of c-AMP. In this group of analogs, the ?i-butyl derivative shows the highest activity on both TSH and GH release. Among the C-8 and C-2 substituted derivatives, the most active on GH release are the 8-bromo- and 8-thio-cAMP compounds. The derivatives with groups in the C-8 position also have a better resistance to phosphodiesterase, Dr. Cehovic says. The increased activity of the new derivatives may be due to one or more mechanisms. The change in a compound's stereochemistry may increase its activity by enabling the compound to better conform to sites on receptor molecules. However, the fact that some derivatives of c-AMP are more resistant to degradation by phosphodiesterase and/or have better cell permeability may be the cause of their greater activity. The exact mechanism of action of these nucleotides is perhaps the most 30 C&EN NOV. 30, 1970
important area of research to be explored, the scientists say. The mechanisms of release of GH and TSH differ from that of PR as indicated by these and other studies. As the exact mechanisms become better known, then perhaps compounds with action even more specific can be synthesized, and control of the hormones will be closer to realization. Derivatives of c-AMP might also be used for control of hormone action at sites other than the pituitary gland. At the New York Academy of Sciences meeting, other scientists explored the role of c-AMP in several of these areas. For example, Dr. Leonard D. Garren, M.D., University of California, San Diego, postulated that the adrenocorticotrophic hormone (ACTH), acting through c-AMP, modulates adrenal protein synthesis by a phosphorylation of the adrenal ribosomes. The ribosomes then synthesize proteins as directed by messenger RNA. These studies have now been confirmed using human adrenal glands, he announced. Reversion. National Cancer Institute scientists George S. Johnson, Robert M. Friedman, and Ira Pastan found in in vitro studies that sarcoma (malignant tumor) cells treated with c-AMP or an analog—6-N-monobutyryl-c-AMP—revert to some morphological and growth characteristics of normal fibroblasts. The NCI group found—as other scientists have f o u n d -
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that action of c-AMP is potentiated with theophylline. The response is quite specific, the researchers point out; ATP, ADP, 5'-AMP, adenosine, adenine, isoproterenol, and epinephrine are ineffective. Dr. John M. Marsh, University of Miami school of medicine, Miami, Fla., reported that c-AMP stimulated progesterone synthesis equal to that of luteinizing hormone (LH) on bovine corpus luteum. The DBC derivative was considerably more active than cAMP, he says. Also, as the NCI scientists found, the effect was specific for c-AMP and DBC, compared to the same or similar compounds used by the NCI group. It is possible, he says, that prostaglandins effect progesterone via c-AMP. Other scientists at the meeting pointed out that cellular response of c-AMP is only activated by the hormone responsible for the action of that cell. For example, c-AMP would not be activated to its second-messenger role if the corpus luteum had been bathed with GH, TSH, or PR hormones. Thus, although c-AMP is involved in many body functions, it must in turn be stimulated by the proper chemical to do a specific messenger service. Much of the chemistry of these actions is still to be discovered. Progress is being made, however, and perhaps one day the cAMP system may be able to be at least partially controlled.
