Constrained Corticotropin-Releasing Factor (CRF) Agonists and Antagonists with <i>i</i>−(<i>i</i>+3) Glu-Xaa-dXbb-Lys Bridges<sup>†</sup>

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an α-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20−23)[dPhe<sup>12</sup>,Glu<sup>20</sup>,Lys<sup>23</sup>,Nle<sup>21,38</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub> and of astressin {cyclo(30−33)[dPhe<sup>12</sup>,Nle<sup>21,38</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub>}, two potent CRF antagonists, and of cyclo(30−33)[Ac-Leu<sup>8</sup>,dPhe<sup>12</sup>,Nle<sup>21</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>,Nle<sup>38</sup>]hCRF<sub>(8</sub><sub>-</sub><sub>41)</sub>, the shortest sequence equipotent to CRF reported to date (Rivier et al. <i>J.</i> <i>Med.</i> <i>Chem.</i> <b>1998</b>, <i>41</i>, 2614−2620 and references therein). To test the hypothesis that the G<u>lu<sup>20</sup>−Ly</u>s<sup>23</sup> and G<u>lu<sup>30</sup>−Ly</u>s<sup>33</sup> lactam rings were favoring an α-helical conformation rather than a turn, we introduced a d-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a d-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a d-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, α-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [dHis<sup>32</sup>] (<b>6</b>) or acetylation of the N-terminus (<b>7</b>) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala<sub>13</sub>-NH<sub>2</sub> scaffold in order to quantify the structural influence of specific l- and dAla<sup>6</sup> and l- and dAla<sup>7</sup> substitutions in [Glu<sup>5</sup>,Lys<sup>8</sup>]Ac-Ala<sub>13</sub>-NH<sub>2</sub> in a standard α-helical configuration. Models of the general form [Glu<sup>5</sup>,lAla<sup>6</sup> or dAla<sup>6</sup>,lAla<sup>7</sup> or dAla<sup>7</sup>,Lys<sup>8</sup>]Ac-Ala<sub>13</sub>-NH<sub>2</sub> were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0−4, 1−5, and 8−12 O−H hydrogen bond donor−acceptor pairs to maintain α-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix−turn−helix motif around a turn centered at residue 31. Such a turn brings Gln<sup>26</sup> in close enough proximity to Lys<sup>36</sup> to suggest introduction of a bridge between them. We synthesized dicyclo(26−36,30−33)[dPhe<sup>12</sup>,Nle<sup>21</sup>,Cys<sup>26</sup>,Glu<sup>30</sup>,Lys<sup>33</sup>,Cys<sup>36</sup>,Nle<sup>38</sup>]Ac-hCRF<sub>(9</sub><sub>-</sub><sub>41)</sub> which showed significant α-helical content using circular dichroism (CD) and had low, but measurable potency {0.3% that of <b>6</b> or ca. 25% that of [dPhe<sup>12</sup>,Nle<sup>21,38</sup>]hCRF<sub>(12</sub><sub>-</sub><sub>41)</sub>}. Since the 26−36 disulfide bridge is incompatible with a continuous α-helix, the postulate of a turn starting at residue 31 will need to be further documented.