PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2010 August 15.
Published in final edited form as:
PMCID: PMC2774256
NIHMSID: NIHMS132484

A comprehensive study of Sansalvamide A derivatives: the structure-activity relationships of 78 derivatives in 2 pancreatic cancer cell lines

Abstract

We report an extensive structure-activity relationship (SAR) of seventy-eight compounds active against two pancreatic cancer cell lines. Our comprehensive evaluation of these compounds utilizes SAR that allow us to evaluate which features of potent compounds play a key role in their cytotoxicity. This is the first report of 19 new second-generation structures, where these new compounds were designed from the first generation of 59 compounds. These 78 structures were tested for their cytotoxicity and this is the first report of their activity against 2 pancreatic cancer cell lines. Our results show that out of 78 compounds, three compounds are worth pursuing as leads, as they show potency of ≥55% in both cancer cell lines. These three compounds all have a common structural motif, 2 consecutive D-amino acids and an N-methyl moiety. Further, of these three compounds, two are second-generation structures, indicating that we can incorporate and utilize data from the first generation to design potency into the second generation. Finally, one analog is in the mid nanomolar range, and has the lowest IC50 of any reported San A derivative. These analogs share no structural homology to current pancreatic cancer drugs, and are cytotoxic at levels on par with existing drugs treating other cancers. Thus, we have established Sansalvamide A as an excellent lead for killing multiple pancreatic cancer cell lines.

1. Introduction

Natural products provide an excellent source of lead structures for new drugs. These novel structures are critical for development of new therapeutic small molecules that target unique biological pathways. Sansalvamide A (San A) is one such natural product (Figure 1). San A, which was isolated from a marine fungus (Fusarium ssp.), exhibits anti-tumor activity on multiple cancer cell lines.13 The natural product is a depsipeptide (Figure 1) and, therefore, prone to ring opening at the ester bond by esterases. Silverman and co-workers found that the natural product peptide (where the hydroxy acid is converted to an amino acid) was 10 fold more active than the natural product depsipeptide.4, 5 Thus, to avoid deactivation via ring-opening all 78 derivatives reported by our laboratory6, 7 were synthesized as the San A peptide derivatives. To date, the synthesis of 89 analogs have been reported, 78 by us and 11 by Silverman et. al.4 All 89 derivatives are referred to as “San A-amide” derivatives. The cytotoxicity of San A-amide derivatives against pancreatic8,4, 5 colon,3, 6, 9, 10 breast, prostate, and melanoma cancers4 clearly indicates San A-amide’s excellent potential as a new therapeutic agent for the treatment of various cancers and supports further exploration of this class of compounds. All 11 of the San A-amide derivates prepared by Silverman and co-workers contain only L-amino acids4, 5, 11, 12 and these demonstrate reasonable potency against the colon cancer cell line HCT-116. They attribute potency against this cell line to the placement of multiple N-methyl moieties on the macrocycle. However, our extensive studies on HCT 11613, 14 and our results reported here for pancreatic cancer cell lines PL45 and BxPC-3 indicate that placement of multiple N-methyl moieties does not generate a potent molecule against these 2 cell lines and that there are other factors that contribute to the molecule’s potency.

Figure 1
Retrosynthetic approach

We report here an extensive structure-activity relationship (SAR) of 78 compounds tested for their cytotoxicity against 2 pancreatic cancer cell lines. This report includes the synthesis and activity of 19 new compounds (second generation) that were designed from the first generation of 59 compounds. These data provide a global view of the San A series and their complex SAR against PL45 and BxPC-3. Two of the three most potent compounds’ structures are second generation compounds and, thus, their potency is reported here for the first time. These three structures clearly point to a common modality, where one N-methyl and 2 consecutively placed D-amino acids are important for potency. Further, one compound, 63, has cytotoxicity in the mid nanomolar region, which is the lowest reported IC50 of any San A derivative.

Peptides are sometimes considered poor drugs for 2 reasons: solubility and rapid degradation within cells. In order for linear peptides to achieve 3-D structures that will bind appropriately to their protein targets, they are often composed of extended sequences of amino acids, making them insoluble. Cyclic peptides, like San A, often perform better as drugs than linear peptides because a small number of amino acids define a rigid 3-D structure. San A-amide analogs also have the advantage that they are lipophilic and therefore have rapid membrane absorption.15 Further, cyclic peptides tend to have greater binding affinity for protein targets than their linear counterparts or other small molecules because they have restricted bond rotation and conformational restraint, which may lock a molecule into an ideal binding mode.16 In addition, cyclic peptides degrade much slower than linear peptides because proteases have difficulty cleaving amide bonds located within a macrocycle.15 Cyclic peptides also have commercially available chemical diversity (i.e. amino acids), are efficiently synthesized, have defined 3-D structures (which is typically required for good binding affinity for protein targets), are effective at penetrating cell membranes, and are stable within cells. To date, there are 720 clinically used peptide drugs or drug candidates: 38% of these are in clinical trials, 56% are in advanced preclinical phases, and 5% are on the market.1719 These peptide drugs are used as prostate and breast cancer antitumor agents, HIV protease inhibitors, osteoporosis-treating drugs, and immunosuppressants.1921 Thus, there is outstanding precedence for treating diseases with peptides.

Pancreatic cancer is the fifth most deadly cancer in the U.S. Only 10% of patients are eligible for surgery22 and less than 20% of pancreatic cancers respond to the current drugs of choice, one of which is Gemcitabine (2,2-difluorodeoxycytidine).23 Although progress has been made on improving survival rates, current therapy is far from acceptable. Indeed, the five-year survival rate for patients with pancreatic cancers is still less than 5%.24 With such a poor rate of response to current chemotherapeutic methods, there is an immediate need for new and effective pancreatic cancer treatments. This work reports the SAR of seventy-eight San A-amide derivatives against two drug-resistant pancreatic cancer cell lines and establishes a structural template that can be used to design new compounds.

2. Results and Discussion

2.1 Synthesis of all compounds

All seventy-eight derivatives described here were constructed as the peptide analogs (Figure 1, where the hydroxy acid was exchanged for an amino acid at position IV). A succinct synthetic protocol has been developed for the creation of these molecules.6, 25 Our compounds have been designed to explore how the placement of N-methyl moieties, D-amino acids, aromatic, and hydrophilic amino acids impact the cytotoxicity of the molecule. These derivatives have logP values between 0.18–3.35 (see supplemental material for a table), thus meeting Lipinski’s rules for solubility and effective diffusion through cellular membranes.26, 27 Our synthetic approach utilized a convergent solution-phase strategy in order to establish a reliable and inexpensive route for the large-scale production of compound needed for additional biological studies (Figure 2). Our outlined route provides rapid and high-yielding synthesis of these compounds.

Figure 2
Synthesis of Macrocycles Conditions: a) coupling agent*, DIPEA (4 equiv), DCM (0.1M), b)TFA (20%), Anisole (2 equiv), DCM, c) LiOH (4 equiv), MeOH d)LiOH (10 equiv), MeOH, e) HATU (0.7 equiv), DEPBT (0.7 equiv), TBTU (0.7 equiv), DIPEA (6 equiv), DMF:DCM:CH ...

The synthesis of the San A-amide derivatives, were completed using the amino acids shown in Figure 3 via the synthetic strategy shown (Figure 2). Using these amino acids, we were able to significantly vary the San A-amide structures in order to explore the role of stereochemistry, N-methyl moieties and aromatic or hydrophilic amino acids in cytotoxicity.

Figure 3
Amino acids used in the synthesis of seventy-eight derivatives

The 78 structures and their cytotoxicity against the 2 pancreatic cancer cell lines are discussed in order of position. That is, structures are arranged in order of amino acids that are altered from the original San A peptide, where alterations to residues in positions I-V are described in Figure 4Figure 8 respectively.

Figure 4
Compounds with alterations at position I. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM compound concentration.
Figure 8
Compounds with alterations at position V and/or I-IV. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM compound concentration. ...

Using 2(1-H-benzotriazole-1-yl)-1,1,3-tetramethyl-uronium tetrafluoroborate (TBTU), and diisopropylethylamine (DIPEA), acid protected residue I (a–f) and N-Boc protected residue II (a–h) were coupled to give the dipeptide I-II-Boc (80–94% yield). Deprotection of the amine on residue II using TFA gave the free amine, I-II (~quantitative yields). Coupling of the dipeptide to monomer III (a–j) gave the desired tripeptide (Fragment 1) in good yields (80%–95%).28 The synthesis of Fragment 2 was completed by coupling residue IV (a–f) to residue V(a–g) to give dipeptide IV-V-Boc (90–95% yield). The amine was deprotected on Fragment 1 using TFA and the acid was revealed in Fragment 2 using lithium hydroxide. Fragment 1 and Fragment 2 were coupled using multiple coupling agents,7, 25, 29, 30 yielding seventy-eight examples of linear pentapeptides (66–90% yield).28

In the case of the di- and tripeptide construction, acid/base workup removed excess reagents and side products, and the NMR indicated compounds did not usually require further purification. Typically, it was only in the case of pentapeptides and macrocycles that we found it necessary to purify compounds via silica chromotography, making the synthesis efficient. The purity of all compounds was verified via NMR and/or LCMS. The linear peptides were then cyclized using similar conditions developed in our laboratory.25 Upon cyclization, the final compounds were purified via flash chromatography and/or HPLC. When appropriate, the side chains were deprotected (Serines, Lysines, and Tyrosines). The purity of all compounds was verified by NMR and LCMS.25

2.2 Structure-activity relationships (SAR)

Although major efforts have been made, few truly novel classes of compounds have shown activity against drug-resistant pancreatic cancer tumors.23, 24 With this in mind, our work elucidates an understanding of the structure-activity relationship (SAR) of seventy-eight San A derivatives against two drug-resistant pancreatic cancer cell lines (PL45, BxPC-3).31 This SAR study describes an overview of the cytotoxicity in cell-based assays for the San A series of molecules and establishes a relationship between the structures and stereochemistry of the active compounds’ amino acid side chains and their growth inhibition against these two cell lines. We also highlight three compounds, two of which are second generation, and are potent (≥55% cytotoxicity) in both cell lines. All three compounds have similar structural motifs: an N-methyl incorporated in 2 consecutive D-amino acids, where one of these residues is a D-Phe. Given that there is an incomplete understanding of the structure-activity relationship (SAR) of San A, we used a medicinal chemistry approach to evaluate which structural features are key to potency. This approach involves synthesizing analogues that have both single and multiple changes relative to the San A natural product peptide and testing them for potency. The core peptide structure (Figure 3) serves as a scaffold and compounds’ potency is organized by position (I-V), highlighting amino acids altered from the original San A-amide pentapeptide macrocycle (Figure 4Figure 8). Cell proliferation was monitored by measuring how much 3H-thymidine was incorporated into a cell’s DNA. This data can be analyzed to determine a particular compound’s activity. 3H-thymidine uptake assays were run using two distinguishable pancreatic cancer cell lines: PL45 and BxPC-3. Data shown gives percent growth inhibition at 5µM concentrations. Only growth inhibition values greater than or equal to 55% are considered “potent” and only those compounds that surpass this threshold in both cell lines are deemed lead structures. The use of two cell lines ensures that our compounds are consistently inhibiting drug-resistant pancreatic cancers and clearly establishes SAR. Potency exhibited by the San A peptide 1 is shown so that comparisons can be made between the natural product peptide and our synthetic analogs.

2.3 SAR Position I

The histogram in Figure 4 shows the percent growth inhibition produced by the addition of compounds (5µM concentration) that have changes to position I against two pancreatic cancer cell lines. Compared to 1, two compounds show a moderate increased potency against PL45 and BxPC-3: Compound 2, which incorporated a D-Phenylalanine, and 4 places a constrained aromatic moiety, an L-tetrahydroisoquinoline amino acid (aa), at position I. However, both compounds are only potent against one cell line, and thus they are not leads. Compounds 5 and 6 show that a H-bonding element (tyrosine and tryptophan respectively) does not improve the cytotoxicity of the molecules over that of San A-amide. In summary, no molecules with a change to position I are considered leads.

2.4 SAR Position II

The histogram in Figure 5 shows the percent growth inhibition for compounds with changes made to position II, where 15 is a second generation structure that also incorporates a change at position I. Compound 7, which incorporates an N-methyl-L-leucine at position II, shows a significant decrease in growth inhibition over that of compound 1. Replacing the L-Leucine with a D-Leucine at II (compound 8) slightly increased potency against PL45 but decreased potency against BxPC-3. However, incorporation of an N-methyl- D-Leucine (compound 9) dramatically decreased the potency against both cell lines. Inclusion of an aromatic moiety (compounds 10, 12, and 14), versus a hydrophilic moiety (compounds 11 and 13) indicates that aromatic residues improve cytotoxicity relative to compounds with polar residues at II (e.g. compare 10 / 12 to 13, and 11 to 14). A new second generation compound, 15, contains 2 aromatic moieties. This molecule is cytotoxic against one cell line, showing ≥55% cytotoxicity against one cell line, but it is poor against a second cell line, as such it is not considered a valuable lead structure.

Figure 5
Compounds with alterations at positions II and/or I. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM compound concentration. ...

2.5 SAR Position III

The histogram in Figure 6 shows the percent inhibition of growth by compounds with changes at position III (some of which also contain changes at positions I and/or II), where compounds 32, 33 and 34 are all second-generation structures. Compound 16 incorporates an N-methyl-valine, compound 17 a D-valine, and compound 18, a N-methyl- D-valine at position III. None show greater than 55% growth inhibition against one cell line. In contrast to 1, which contains an isopropyl moiety at position III, compounds containing a methyl 19 or an ethyl 20 moiety were tested and showed significantly reduced potency against both cell lines. Compound 21 contains an ethyl moiety at position III along with a D-Leucine at position II and shows lower cytotoxicity than 8, which is its “parent” compound. Interestingly, placing either a hydrophilic element or an aromatic element at position III, 22/24 or 23/25 respectively, does not lead to increased potency against either cell line. Compound 26 and 27 share an almost identical core structure, with the same residues in positions I, II, IV, and V, but 26 has an D-Valine and 27 has D-ethyl glycine at III. Both compounds are below 55% against both cell lines. Similarly 28 is also not potent. Compound 29, which incorporates a D-Phe at I, a D-Leu at II, and a D-Valine at III, shows some potency against both cell lines, but under the 55% required to make this molecule a lead. Compounds 30 and 31 involved alterations at both positions II and III, and both have potency below 55% in each cell line. However, a new second generation compound, 32, exhibited improved cytotoxicity against PL45 relative to both its starting molecules, 12 and 17, but not against both cell lines. Comparing 32 to 33 and 34 show that the incorporation of an N-methyl and 2 non-consecutively placed D-amino acids is not enough to produce compounds with ≥55% cytotoxicity in PL45.

Figure 6
Compounds with alterations in positions III and/or I/II. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM compound concentration. ...

2.6 SAR Position IV

The histogram in Figure 7 shows the percent inhibition of growth by compounds with changes to position IV (where some also include changes to position(s) I, II and/or III). Compounds 37, 42, 43, 47, and 49 are all second generation structures. In contrast to potent compound 17, which has a D-aa at III, 35 (D-aa at position IV), shows 0% inhibition against both cell lines. Further, the derivative with the N-methyl at III is non-potent, whereas compound 36, which incorporates an N-methyl moiety at IV, showed reasonable inhibition against both cell lines. Compound 37, which has 2 D-amino acids consecutively placed at III and IV, was not potent; neither was 38, which has D-aa substitutions at positions I, III, and IV. Compounds 39, 40, 41, 42, and 43 all contain a lysine at position IV, as well as other changes at various other positions. All five compounds are uniformly poor growth inhibitors against both cell lines. Not surprisingly, the hydrophobic compounds that are structurally related to these polar compounds, 44, 45, 46, and 47 respectively, are also relatively non-toxic and only show a low level of potency against these cell lines. Further, the incorporation of an N-methyl D-Valine at III, and a cyclohexyl moiety at position IV produced compound 48, which showed improved cytotoxicity against both cell lines relative to its parent (18). Finally, second generation compound 49, exhibited underwhelming cytotoxicity against both cell lines.

Figure 7
Compounds with alterations at positions IV and/or l-lll. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM compound concentration. ...

2.7 SAR Position V

The histogram in Figure 8 shows the percent inhibition of growth by compounds with changes at position V (where some also include changes at position(s) I, II, III and/or IV), where compounds 60, 61, 62, 63, 64, 65, 66, 67, 68, and 69 are all second generation compounds. Compound 50 incorporates an N-methy-L-leucine at position 5, compound 51 a D-leucine, and compound 52 an N-methyl D-leucine. Both 50 and 52 exhibit good growth inhibition against both cell lines but are not ≥55%. Interestingly 53, which contains an N-methyl-L-Leucine at position IV and D-Leucine at position V, showed some promise in one cell line. Similar to previous observations, the inclusion of a D-Phe (at I) made 54 a promising lead structure. Incorporating a D-Leucine at positions IV and V gave non-toxic 55. Not surprisingly compounds 56 and 57, which contain a D-Leucine at position V and are structurally similar to non-potent compound 51, showed poor growth inhibition against both cell lines. Interestingly, compounds 57 and 58 have reduced potency compared to their parent compounds 1, 17, 50, or 51. Compound 60 has a polar residue, and as noted earlier, this property makes the molecule not effective. 61 and 62 are both relatively non-toxic; yet 63 shows reasonable potency in both cell lines, with cytotoxicity values of ≥55%. Similar to 54, compound 63, has 2 consecutive D-amino acids and an N-methyl moiety. Consistent with previous observations, polar derivative 64 is not toxic, nor are derivatives 68 and 69. Comparison of compound 65 and 66 indicates that two consecutively placed D-amino acids play an important role in the potency of 66, where 66 is also considered a lead. Compound 67 is based on 29 (Figure 6), yet it does not perform as well as 29. In summary, three compounds, two being second generation, show greater than 55% in each cell line: 54, 63, and 66.

2.8 SAR all L and D amino amino acids

The histogram in Figure 9 shows the percent inhibition of growth by compounds that contain all L or all D-amino acids. Compound 70 is the enantiomer of 1, and its cytotoxicity is lower than that of 1. Compound 71 is the enantiomer of 7 and, in addition to all D-amino acids, it contains an N-methyl at position II. Although it is more potent than 7, it is not as potent as 1. Compound 16 and 72 are enantiomers with an N-methyl at position III, and both are non-potent. Compound 50 with an N-methyl at V is potent and interestingly its enantiomer 75 maintains relatively similar potency. Enantiomers 73 and 74, which contain 2 N-methyl moieties (positions II and V), are also not active. The same is true for enantiomers 76 and 77, which also contain 2- N-methyl moieties (positions III and V). Finally compound 78 with three N-methyls is not active. Thus, this series of molecules has no compounds with ≥ 55% cytotoxicity in any cell line.

Figure 9
Compounds with N-methyl moieties and their enantiomers. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. Error = ±5%. All assays were run for 72 hours at 5µM ...

2.9 Design of potent compounds

Shown in Figure 10 are the three most potent compounds: 54, 63, and 66 and the compounds from which they were designed. Two of these three compounds are reported here for the first time. It is important to note that all three of these compounds have an N-methyl associated with 2 consecutive D-amino acids, where one of these is a D-Phenylalanine. In every case one can see that the final compound showed an average improved potency in both cell lines over the compounds used to rationally design them. 54 was designed from the combination of several compounds: 2, 36, and 53, and 63 was designed from compound 54. Finally 66 was designed from 12, 17, and 48.

Figure 10
Most potent compounds and the compounds from which they were designed. Data is represented as % growth inhibition relative to 1% DMSO control. Each data point is an average of 4 wells run in three assays. All assays were run for 72 hours at 5µM ...

Compound 54, which contains a N-Methyl at IV and D-amino acids at positions I and V, was designed from compounds 2, 36, and 53, where 2 contained a D-amino acid at I, 36 an N-methyl at IV, and 53 had both a D-amino acid at V and an N-methyl at IV. Thus, although these three compounds, 2, 36, and 53 showed some potential, 54 shows increased potency for both cell lines over that shown by the three initial compounds from which they were designed. Designed from 54 (this can be perceived by rotating 54 clockwise by one position), 63 maintained the motif of an N-methyl followed by two consecutive D-amino acids. Its improved cytotoxicity over that of 54 may result from a large hydrophobic moiety placed at position IV or the addition of another aromatic moiety at position 1. Compound 66 was designed from compounds 12, 17, and 48 where it is comprised of an N-methyl D-Phe at II (from 12), a D-Val at III (from 17), a cyclohexyl moiety at IV (from 48), and the addition of a benzyl protected serine ether at V. Thus, 66 has three aromatic moieties placed around its macrocyclic backbone and it is significantly more potent than its precursors. It is interesting to note that several other derivatives, specifically 15 and 32, also contain 2 consecutive D-amino acids and an N-methyl moiety, yet these 2 compounds are only cytotoxic against PL45, and in fact show very poor cytotoxicity against BxPC-3. 15 has a tyrosine at I, and thus, this may impact its cytotoxicity. 32 is structurally similar to 66, but it lacks the hydrophobic moieties at IV and V, which, as noted with 63, appear to play an important role in the generating a cytotoxic molecule.

In summary, we report here for the first time 3 potent compounds out of 78 derivatives, where potency was designed into our compounds using known structural features that increase their cytotoxicity i.e. N-methyls and D-amino acids. We successfully generated compounds that show consistency across both pancreatic cancer cell lines, and have devised a general approach to generating improved derivatives.

2.10 IC50 Determination

Inhibition constants were calculated for the three most potent compounds by plotting five concentrations (50, 10, 0.5, and 0.1µM) and extracting data from the curves (supplementary data). All relationships are logarithmic in nature. We have identified 2 compounds, 54 and 66, that show low micromolar cytotoxicity and 1 compound, 63, that shows mid nanomolar cytotoxicity against pancreatic cancer cell lines. The IC50 values for the most potent compounds are shown in Figure 11, where these compounds show up to ~40 fold greater cytotoxicity than the parent natural product peptide 1. It is interesting to note that although 63 does not display the highest cytotoxicity values against the 2 cell lines at 5µM, it does show by far the best IC50 value, 500nM in PL45. This appears to result from the 5µM data point residing (consistently) lower than the curve. Thus, overall we have found a single compound (63) with a nanomolar IC50 value against these drug resistant cancer cell lines, as well as determined that 66 shows potential as a lead. Both compounds are new derivatives, reported here for the first time.

Figure 11Figure 11
IC50s of potent compounds. Each data point is an average of 4 wells run in three assays at 50, 10, 0.5, and 0.1 µM.

2.11 Summary of SAR results

The most important feature to emerge from this SAR study is the observation that molecules that are potent contain 2 consecutive D-amino acids and an N-methyl located next to the D-amino acids. Indeed, all potent compounds contain 2 consecutive D-amino acids, one of which is a D-Phenylalanine, and an N-methyl on or next to the D-amino acids. It is easy to recognize this pattern by comparing the potency of 2, 36, and 53 to 54 and that of 12, 17, and 48 to 66. Neither of the parent compounds are as cytotoxic as 54 and 66 respectively.

Further, comparison of the potency of 65 to 66 clearly indicates that 2 D-amino acids next to each other significantly improve the compound’s cytotoxicity. Further validating our hypothesis is comparison of compounds 30 and 31, where 30 contains 2 D-amino acids sequentially placed, and 31 contains 2 N-methyl D-amino acids successively placed. Both have low cytotoxicity relative to our lead compounds. Thus, it is not enough to have 2 sequential D-amino acids, nor 2 N-methyl D-amino acids to generate a lead structure. Rather, as observed by others in this pentapeptide system, D-amino acids play an important role in achieving a favorable conformation.3234

Finally, it is important to note that structurally similar compound 56 contains 2 D-amino acids and an N-methyl but the D-amino acids are not consecutive, and its potency is poor relative to that of 54 or 66. Also note that 34 is not nearly as potent as other compounds with consecutive D-amino acids. Finally, more than one N-methyl and/or more than 2 consecutively placed D-amino acids are not potent (i.e. 61, 76, 77, and 78).

In summary, all compounds found to exhibit cytotoxicity that was equal to or greater than 55% in each pancreatic cancer cell line contained a single N-methyl and 2 sequentially placed D-amino acids. As such, we surmise that two factors are important for potency: a single N-methyl in conjunction with 2 consecutively placed D-amino acids, where one is a D-Phenylalanine. This theory is validated by several current examples described in the recent literature, where cyclic peptides, specifically pentapeptides, with D-amino acids lock the macrocycle into a specific conformation.3234 Further, it is well established that these cyclic pentapeptides mimic beta and gamma turns and serve as templates for the appropriate positioning of suitable binding motifs for proteins.35, 36 Thus, we believe that the inclusion of 2 successive D-amino acids coupled with a single N-methyl locks the macrocycle into a suitable position for binding to its biological target.

3. Conclusion

We report here for the first time a comprehensive SAR evaluation using two generations of compounds, where 19 new structures and 59 previously published structures were described and examined for their cytotoxicity against two drug-resistant pancreatic cancer cell lines. This global assessment included the determination that placement of a single N-methyl and 2 consecutively placed D-amino acids are required for compounds to exhibit ≥55% cytotoxicity in both cell lines, where one compound exhibited a 500 nM IC50. Two of the three most potent compounds were second-generation compounds, and these two compounds had improved IC50s over the first generation structure. Our three most potent small molecules target drug-resistant pancreatic cancer cell lines, and share no homology with other classes of chemotherapeutic agents and have reasonable clog P values37 and molecular weights (600–738). Studies on third generation compounds are on going and will be reported in due course.

4. Experimental Procedures

4.1 General remarks

All coupling reactions were performed under argon atmosphere with the exclusion of moisture. All reagents were used as received. Anhydrous methylene chloride Dri Solv (EM), anhydrous tetrahydrofuran, Andydrous dimethylformamide, and anhydrous acetonitrile Dri Solv (EM) were obtained from VWR, and were packed under nitrogen with a septum cap. Diisopropylethylamine (DIPEA) was purchased from Aldrich, packaged under nitrogen in a sure seal bottle. The coupling agent HATU came from: Applied Biosystems at 850 Lincoln Center Dr. Foster City, CA 94404, USA. Tel.: +1-800-327-3002 and the coupling agents TBTU NovaBiochem. DEPBT [3(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)] was purchased from Aldrich (order number 49596-4). The 1H NMR spectra were recorded on the Varian at 200MHz or 400MHz or 500 MHz. LCMS was performed at San Diego State University using HP1100 Finnigan LCQ. Flash column chromatography used 230–400 mesh 32–74 Im 60Å silica gel from Bodman Industries.

4.2 Thymidine Incorporation/Growth inhibition assay

Proliferation of the PL-45 and BxPC-3 pancreatic cancer cells was tested in the presence and absence of the compounds using 3H-thymidine uptake assays. Cells treated with the compounds were compared to DMSO controls for their ability to proliferate as indicated by the incorporation of 3H-thymidine into their DNA. Cells were cultured in 96 well plates at a concentration of 2000–2500 cells/well in DMEM (Gibco) supplemented with L-glutamine, 10% fetal bovine serum and 1% penicillin-streptomycin antibiotic. After incubation for 6 h, the compounds were added. The compounds were dissolved in DMSO at a final concentration of 1% and tested at the concentrations indicated in the manuscript. The DMSO control was also at 1%. After the cells had been incubated with the compounds for 56 h, 1mCi 3H-thymidine per well was added and the cells were cultured for an additional 16 h (for the cells to have a total of 72 h treatment), at which time the cells were harvested using a PHD cell harvester (Cambridge Technology Inc.). The samples were then counted (CPM) in a scintillation counter for 5 m. Decreases in 3H-thymidine incorporation, as compared to DMSO controls, are an indication that the cells are no longer progressing through the cell cycle or synthesizing DNA, as is shown in the studies presented. Mean growth inhibition (n=8–12) is the 1 minus CPM of compound-treated cells over DMSO-treated cells. IC50 were determined using 0, 0.1, 0.5, 5, 10, and 50 µM of compound. All calculations including mean, SEM, and IC50 were performed on Excel.

4.3 Synthesis

For synthesis details of the first-generation compounds see reference 6. For second-generation compounds see reference 7. Potent compounds’ final characterization data are listed below.

4.3.1 General peptide synthesis

All peptide coupling reactions were carried out under argon with dry solvent, using methylene chloride for dipeptide and tripeptide couplings and a mixture of dimethylformamide, methylene chloride and acetonitrile for pentapeptide couplings. The amine (1.1 equivalents) and acid (1 equivalent) were weighed into a dry flask along with 4–12 equivalents of DIPEA and 1.1 equivalents of TBTU.* Anhydrous methylene chloride was added to generate a 0.1M solution. The solution was stirred at room temperature and reactions were monitored by TLC. Reactions were run for 1 hour before checking via TLC. If reaction was not complete additional 0.25 equivalents were of HATU and TBTU were added. If reaction was complete then work-up was done by washing with aqueous HCl (pH 1) and saturated sodium bicarbonate. (Note: if acetonitrile was used for the reaction, methylene chloride was added to reaction upon workup and the resulting solution was washed with the aqueous solvents). After back extraction of aqueous layers with methylene chloride, organic layers were combined, dried over sodium sulfate, filtered and concentrated. Flash chromatography using a gradient of ethyl acetate-hexane gave our desired peptide.

*Some coupling reactions would not go to completion using only TBTU and therefore ~0.25 equivalents of HATU, and/or DEPBT were used. In a few cases up to 1.7 equivalents of all three coupling reagents were used.

4.3.2 General Amine deprotection

Amines were deprotected using 20% TFA in methylene chloride (0.1M) with two equivalents of anisole. The reactions were monitored by TLC, where the TLC sample was first worked up in a mini-workup using DI water and methylene chloride to remove TFA. Reactions were allowed to run for 1–2 hours and then concentrated in vacuo.

4.3.3 General Acid deprotection

Acids were deprotected using ~4 equivalents of lithium hydroxide (or until pH=~11) in methanol (0.1 M). The peptide was placed in a flask, along with lithium hydroxide and methanol and stirred overnight. Within 12 hours the acid was usually deprotected. Work-up of reactions involved the acidification of reaction solution using HCl to pH = 1. The aqueous solution was extracted three times with methylene chloride, and the combined organic layer was dried, filtered and concentrated in vacuo.

4.3.4 Macrocyclization procedure (in situ)

All pentapeptides were acid and amine deprotected using concentrated HCl (8 drops per 0.3mmols of linear pentapeptide) in THF (0.05M). Anisole (2 equivs) was added to the reaction and the reaction was stirred at room temperature. The reaction typically took 4 days, but TLC and LCMS were used to monitor the reaction every 12 hours. LCMS data typically indicated the reaction was ~50% complete after the first day. Addition of four drops of concentrated HCl per 0.3 mmol of pentapeptide, stirring at RT overnight and checking the reaction via LCMS usually showed ~75% completion. On the fourth day verification of the presence of the free amine and free acid and disappearance of the starting linear protected pentapeptide permitted workup. The reaction was concentrated in vacuo and the crude, dry, double deprotected peptide (free acid and free amine) was dissolved in a minimum solution of DMF: methylene chloride: acetonitrile (2:2:1 ratio). Three coupling agents (DEPBT, HATU, and TBTU) were used at ~0.5 to 0.75 equivalents each. These coupling agents were dissolved in a calculated volume of dry 40% DMF, 40% methylene chloride, and 20% acetonitrile that would give a 0.01 M overall solution when included in the volume used for the deprotected peptide. The coupling agents were then added to the deprotected peptide solution. DIPEA (6 equivs or more in order to neutralize the pH) were then added to the reaction. The coupling agents are typically not very soluble in acetonitrile, which is why a combination of solvents is used.

After 1 hour, TLC and LCMS (where the LCMS sample was worked up prior to injection) indicated that a product spot was developing. The comparison Rf value in the product spot on TLC was the protected linear pentapeptide. The reactions were always complete after 3 hours, and monitoring the starting material deprotected pentapeptide via LCMS was the easiest method of determining completion. Upon completion, the reaction was worked up by washing with aqueous HCl (pH 1) and saturated sodium bicarbonate. After back extraction of aqueous layers with large quantities of methylene chloride, the organic layers were combined, dried, filtered and concentrated. All macrocycles were purified by initially running a crude plug of compound using an ethyl acetate/hexane gradient on silica gel, then running a column on the isolated product. Finally, when necessary reverse phase HPLC was used for additional purification using a gradient of acetonitrile and DI water with 0.1% TFA.

4.3.5 Synthesis of Compound 15

Dipeptide

MeO-Val-Leu-NHBoc

Dipeptide MeO-Val-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 370 mg (2.2 mmols, 1.1 equiv.) of amine OMe-Val-NH2, 500 mg (2.0 mmols, 1.0 equiv.) of acid HO-Leu-NHBoc, 1.4 mL (8 equivalents) of DIPEA, 515.2 mg (1.6 mmols, 0.8 equiv.) of TBTU, 305 mg (0.8 mmols, 0.4 equiv.) The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (625mg, 90% yield).

Rf: 0.5 (EtOAc: Hex 1:2)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.6–1.8 (m, 2H), 2.1–2.3 (m, 1H), 3.7 (s, 3H), 4.0–4.1 (m, αH), 4.4–4.5 (m, αH), 4.8–4.9 (br, 1H), 6.6 (d, 1H)

Dipeptide

MeO-Val-Leu-NH2

Dipeptide MeO-Val-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (446mg, 100% yield).

Tripeptide

MeO-Val-Leu-Leu-NHBoc

Tripeptide MeO-Val-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 689 mg (2.82 mmols, 1.0 equiv.) of amine MeO-Val-Leu-NH2, 774 mg (3.11 mmols, 1.0 equiv.) of acid HO-Leu-NHBoc, 1.9 mL (4 equiv.) of DIPEA, 544 mg (1.69 mmols, 0.6 equiv.) of TBTU, 644 mg (1.69 mmols, 0.6 equiv.) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (887mg, 60% yield).

Rf: 0.5 (EtOAc: Hex 1:1) 1H NMR (200 MHz, CDCI3): δ 0.8–1.1 (m, 12H), 1.4 (s, 9H), 2.8 (s, 3H), 3.7 (s, 3H), 4.0–4.2 (m, αH), 4.4–4.6 (m, 2αH), 4.9 (d, 1H), 6.5–6.7 (d, 2H)

Dipeptide

MeO-D-Tyr-N-Me-D-Phe-NBoc

Dipeptide MeO-D-Tyr-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 318 mg (1.4 mmols, 1.1 equiv.) of amine MeO-D-Tyr-NH2, 576 mg (1.3 mmols, 1.0 equiv.) of acid HO-N-Me-D-Phe-NBoc, 1.4 mL (8 equiv.) of DIPEA, 515.2 mg (1.6 mmols, 0.8 equiv.) of TBTU, 380 mg (0.8 mmols, 0.6 equiv.) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (625mg, 90% yield).

Rf: 0.5 (EtOAc: Hex 1:2)

1H NMR (200 MHz, CDCI3): δ 1.4(m, 9H), 2.8 (s, 3H), 2.9–3.4 (m, 4H), 3.7 (s, 3H), 4.7–4.9 (m, 2αH), 6.0 (br, 1H), 6.2 (br, 1H), 6.7–7.0 (dd, 4H), 7.1–7.3 (m, 5H).

Dipeptide

MeO-D-Tyr-N-Me-D-Phe-NH

Dipeptide MeO-D-Tyr-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (625mg, 100% yield).

Dipeptide

HO-Val-Leu-Leu-NHBoc

Dipeptide HO-Val-Leu-Leu-NHBoc was synthesized following the “General acid deprotection”. This tripeptide was taken on to the next reaction without further purification or characterization. (661mg, 88% yield).

Pentapeptide

MeO-D-Tyr-N-Me-D-Phe-Val-Leu-Leu-NHBoc

Pentapeptide MeO-D-Tyr-N-Me-D-Phe-Val-Val-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 503.4 mg (1.41 mmols, 1.0 equivalents) of amine Meo-D-Tyr-D-MePhe-NH, 689 mg (1.55 mmols, 1.0 equiv.) of acid HO-Val-Leu-Leu-NHBoc, 0.86 mL (3.5 equiv.) of DIPEA, 91 mg (0.28 mmols, 0.2 equiv.) of TBTU, and 322 mg (0.85 mmols, 0.6 equiv.) HATU, 169 mg (0.56 mmols, 0.4 equiv.) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (727mg, 60% yield).

Rf: 0.5 (EtOAc: Hex 2:1)

1H NMR (400 MHz, CDCI3): δ 0.6–0.8 (m, 6H), 0.8–1.0 (m, 12H), 1.5–1.8 (m, 5H), 2.6 (m, 2H), 2.8–3.4 (m, 4H), 3.7 (s, 3H), 4.2 (m, αH), 4.4–4.5 (m, αH), 4.5–4.6 (m, αH), 4.6–4.7 (m, αH), 4.7–4.8 (m, αH), 5.0 (d, 1H), 6.6–6.8 (br, 2H), 6.7–7.4 (m, 9H)

Macrocycle

D-Tyr-N-Me-D-Phe-Val-Leu-Leu

Macrocycle D-Tyr-N-Me-D-Phe-Val-Leu-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 182.4 mg (0.27 mmols, 1.0 equivalents) of linear pentapeptide, 0.19 mL (4 equivalents) of DIPEA, 52.6 mg (0.16 mmols, 0.6 equivalents) of TBTU, 41.5 mg (0.11 mmols, 0.4 equivalents) HATU, and 16.3 mg (0.11 mmols, 0.4 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (18mg, 10% yield).

Rf: 0.5 (EtOAc: Hex 4:1)

1H NMR (400 MHz, CDCI3): δ 0.6–1.0 (m, 18H), 1.6–1.8 (m, 4H), 1.8–2.0 (m, 1H), 2.9 (s, 3H), 2.6–3.2(m, 4H), 4.1 (m, αH), 4.2 (m, αH), 4.3–4.5 (m, αH), 4.6–4.7 (m, αH), 5.5 (m, αH), 6.7–7.0 (m, 4H), 7.1–7.4 (m, 5H)

LCMS: m/z calcd for C33H51N5O5 (M+1) = 598.39, found 598.3

4.3.6 Synthesis of Compound 32

Dipeptide

MeO-Phe-N-Me-D-Phe-NBoc

Dipeptide MeO-Phe-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 509 mg (1.51 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 600 mg (1.37 mmols, 1.0 equivalents) of acid HO-N-Me-D-Phe-NBoc, 1.5 mL (4 equivalents) of DIPEA, 828 mg (2.58 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (520.1 mg, 86% yield).

Rf: 0.7 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 1.4 (s, 9H), 2.7 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), 4.8–5.0 (m, 2αH), 7.1–7.3 (dd, 10H)

Dipeptide

MeO-Phe-N-Me-D-Phe-NH

Dipeptide MeO-Phe-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (521 mg, 100% yield).

Tripeptide

MeO-Phe-N-Me-D-Phe-D-Val-NHBoc

Tripeptide MeO-Phe-N-Me-D-Phe-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 521 mg (1.5 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-NH, 302.3mg (1.39 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 0.97 mL (4 equivalents) of DIPEA, 268 mg (0.83 mmols, 0.6 equivalents) of TBTU, and 317 mg (0.83 mmol, 0.6 equivalents) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (316 mg, 45% yield).

Rf: 0.3 (EtOAc: Hex 3:7)

1H NMR (200 MHz, CDCI3): δ 0.5 (d, 6H), 1.4 (s, 9H), 2.9 (s, 3H), 3.1 (t, 4H), 3.7 (s, 3H), 4.1 (m, αH), 4.8 (m, αH), 5.0 (d, 1H), 5.5 (m, αH), 7.2–7.3 (dd, 10H)

Tripeptide

MeO-Phe-D-MePhe-D-Val-NH2

Tripeptide MeO-Phe-N-Me-D-Phe-D-Val-NH2 was synthesized following the “General amine deprotection”. This tripeptide was taken on to the next reaction without further purification or characterization. (316 mg, 100% yield).

Dipeptide

MeO-Leu-Leu-NHBoc

Dipeptide MeO-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 401 mg (2.2 mmols, 1.1 equivalents) of amine MeO-Leu-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (4 equivalents) of DIPEA, 774 mg (1.2 mmols, 1.0 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (703 mg, 98% yield).

Rf: 0.35 (EtOAc: Hex 2.5:7.5)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.6–1.8 (m, 6H), 3.7 (s, 3H), 4.0–4.1 (m, αH), 4.5–4.7 (m, αH), 4.8–4.9 (br, 1H), 6.4 (d, 1H)

Dipeptide HO-Leu-Leu-NHBoc

Dipeptide HO-Leu-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (349 mg, 58% yield).

Pentapeptide

MeO-Phe-N-Me-D-Phe-D-Val-Leu-Leu-NHBoc

Pentapeptide MeO-Phe-N-Me-D-Phe-D-Val-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 316 mg (0.72 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-D-Val-NH2, 240 mg (0.65 mmols, 1.0 equivalents) of acid HO-Leu-Leu-NHBoc, 0.92 mL (8 equivalents) of DIPEA, 126 mg (0.39 mmols, 0.6 equivalents) of TBTU, 174 mg (0.45 mmols, 0.7 equivalents) HATU, and 78 mg (0.26 mmols, 0.4 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (172mg, 34% yield).

Rf: 0.3 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.7–0.9 (m, 18H), 1.4 (s, 9H), 1.4–1.6(m, 2H), 2.0–2.2 (m, 4H), 2.8 (m, 1H), 3.0 (m, 1H), 3.0–3.2 (m, 4H), 3.3 (s, 3H), 3.7 (s, 3H), 4.0–4.1 (m, αH), 4.3–4.4 (m, αH), 4.6–4.7 (m, αH), 5.1 (m, αH), 5.3 (m, αH), 7.1–7.3 (m, 10H)

Macrocycle

Phe-N-Me-D-Phe-D-Val-Leu-Leu

Macrocycle Phe-N-Me-D-Phe-D-Val-Leu-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 139 mg (0.21 mmols, 1.0 equivalents) of linear pentapeptide, 0.3 mL (8 equivalents) of DIPEA, 41 mg (0.13 mmols, 0.6 equivalents) of TBTU, 65 mg (0.17 mmols, 0.8 equivalents) HATU, and 13 mg (0.04 mmols, 0.2 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) yield the macrocycle (2.3 mg, 0.07% yield).

Rf: 0.3 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD30D): δ 0.8–1.0 (m, 18H), 1.3 (m, 2H), 4.4–1.6 (m, 4H), 2.6 (m, 1H), 3.2–3.4 (m, 7H), 4.2 (m, αH), 4.6 (m, αH), 5.0 (m, 3αH), 7.2 (dd, 10H) LCMS: m/z calcd for C36H51N5O5 (M+1) = 634.82, found 634.5

4.3.7 Synthesis of Compound 33

Dipeptide

MeO-D-Phe-Leu-NHBoc

Dipeptide MeO-D-Phe-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 951 mg (4.4 mmols, 1.1 equivalents) of amine MeO-D-Phe-NH2, 1000 mg (4.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 2.8 mL (4 equivalents) of DIPEA, 1545 mg (4.8 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (1381mg, 88% yield).

Rf: 0.3 (EtOAc: Hex 3:7)

1H NMR (200 MHz, CDCI3): δ 0.95 (d, 6H), 1.4 (s, 9H), 1.6–1.8 (m, 3H), 3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.1 (m, αH), 4.8–5.0 (m, αH), 6.4–6.6 (d, 1H), 7.1–7.4 (m, 5H)

Dipeptide

MeO-D-Phe-Leu-NH2

Dipeptide MeO-D-Phe-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (1029mg, 100% yield).

Tripeptide

MeO-D-Phe-Leu-D-Val-NHBoc

Tripeptide MeO-D-Phe-Leu-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 343 mg (1.17 mmols, 1.1 equivalents) of amine MeO-D-Phe-Leu-NH2, 230.4mg (1.06 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 1.49 mL (8 equivalents) of DIPEA, 408 mg (1.25 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (443.8mg, 85% yield).

Rf: 0.7 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.5 (s, 12H), 1.6–1.8 (m, 3H), 2.2 (m, 2H), 3.1(m, 2H), 3.7 (s, 3H), 3.9–4.0 (dd, 1H), 4.4 (m, αH), 4.9 (dd, αH), 5.0 (m, αH), 6.3 (d, 1H), 6.6 (m, 1H), 7.0–7.4 (m, 5H)

Tripeptide

MeO-D-Phe-Leu-D-Val-NH2

Tripeptide MeO-D-Phe-Leu-D-Val-NH2 was synthesized following the “General amine deprotection”. This tripeptide was taken on to the next reaction without further purification or characterization. (353.5mg, 100% yield).

Dipeptide

MeO-Leu-Leu-NHBoc

Dipeptide MeO-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 401 mg (2.2 mmols, 1.1 equivalents) of amine MeO-Leu-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (4 equivalents) of DIPEA, 774 mg (1.2 mmols, 1.0 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (627.4mg, 87.2% yield).

Rf: 0.35 (EtOAc: Hex 2.5:7.5)

1H NMR (400 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.6–1.8 (m, 6H), 3.7 (s, 3H), 4.0–4.1 (br, αH), 4.5–4.7 (m, αH), 4.8–4.9 (br, 1H), 6.4 (d, 1H)

Dipeptide

HO-Leu-Leu-NHBoc

Dipeptide HO-Leu-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (571.8mg, 91% yield).

Pentapeptide

MeO-D-Phe-Leu-D-Val-Leu-Leu-NHBoc

Pentapeptide MeO-D-Phe-Leu-D-Val-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 443.8 mg (1.1 mmols, 1.1 equivalents) of amine MeO-D-Phe-Leu-D-Val-NH2, 354 mg (1. mmols, 1.0 equivalents) of acid HO-Leu-Leu-NHBoc, 1.44 mL (8 equivalents) of DIPEA, 198 mg (0.62 mmols, 0.6 equivalents) of TBTU, and 235 mg (0.62 mmols, 0.6 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (545mg, 73% yield).

Rf: 0.6 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.7–0.9 (m, 24H), 1.4 (s, 9H), 1.5–1.7 (m, 6H), 2.2(m, 2H), 3.0(m, H), 3.1 (m, H), 3.7 (s, 3H), 4.0–4.2 (m, 2αH), 4.3–4.5 (m, 2αH), 4.6–4.7 (m, H), 7.0–7.4 (m, 5H)

Macrocycle

MeO-D-Phe-Leu-D-Val-Leu-Leu-NHBoc

Macrocycle D-Phe-Leu-D-Val-Leu-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 226 mg (0.37 mmols, 1.0 equivalents) of linear pentapeptide, 0.67 mL (10 equivalents) of DIPEA, 96.04 mg (0.3 mmols, 0.8 equivalents) of TBTU, 113.7 mg (0.3 mmols, 0.8 equivalents) HATU, and 44.8 mg (0.15 mmols, 0.4 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) yield the macrocycle (1 mg, 0.5 % yield).

Rf:0.3(100%EtOAc)

1H NMR (500 MHz, CD30D): δ 0.7–0.9 (m, 24H), 1.2–1.7 (m, 9H), 2.0–2.1(m, H), 2.2(m, 1H), 3.0(m, H), 3.1 (m, H), 3.9 (d, αH), 4.0 (dd, αH), 4.4–4.6 (m, 2αH), 5.2(t, H), 7.0–7.4 (m, 5H)

LCMS: m/z calcd for C32H51N5O5 (M+1) = 586.78, found 586.6

4.3.8 Synthesis of Compound 34

Dipeptide

MeO-D-Phe-Leu-NHBoc

Dipeptide MeO-D-Phe-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 480 mg (2.2 mmols, 1.1 equivalents) of amine MeO-D-Phe-NH2, 505 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (4 equivalents) of DIPEA, 772 mg (2.4 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (786mg, 94% yield).

Rf: 0.9 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m, 2H), 1.8–2.0 (m, 1H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br, 1H), 4.8 (br, αH), 4.8–5.0 (q, αH), 6.5 (m, 1H), 7.1–7.4 (m, 5H)

Dipeptide

HO-D-Phe-Leu-NHBoc

Dipeptide HO-D-Phe-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (632mg, 89% yield).

Dipeptide

MeO-Leu-Leu-NHBoc

Dipeptide MeO-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 801 mg (4.4 mmols, 1.1 equivalents) of amine MeO-Leu-NH2, 1.0 g (4.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 2.8 mL (4 equivalents) of DIPEA, 1.5 g (4.8 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (1.4 g, 99% yield).

Rf: 0.9 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.6–1.8 (t, 6H), 3.7 (s, 3H), 4.0–4.1 (dd, αH), 4.5–4.7 (m, αH), 4.8–4.9 (br, 1H), 6.4 (d, 1H)

Dipeptide

HO-Leu-Leu-NHBoc

Dipeptide HO-Leu-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (1.2 g, 82% yield).

Tripeptide

MeO-N-Me-D-Val-Leu-Leu-NHBoc

Tripeptide MeO-N-Me-D-Val-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 258 mg (2.3 mmols, 1.1 equivalents) of amine MeO-N-Me-D-Val-NH, 563 mg (1.6 mmols, 1.0 equivalents) of acid HO-Leu-Leu-NHBoc, 1.1 mL (4 equivalents) of DIPEA, 620 mg (1.6 mmols, 1.0 equivalents) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (341 mg, 40% yield).

Rf: 0.5 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.9–1.1 (m, 18H), 1.5 (s, 9H), 1.6–1.8 (m, 1H), 2.1 (m, 2H), 2.8 (s, 1H), 3.0 (d, 3H), 3.7 (s, 3H), 4.0–4.2 (br, 2H), 4.2 (m, αH), 5.0 (s, αH), 5.1 (s, αH), 5.2–5.4 (br, αH), 5.5 (m, 1H), 6.8 (d, 1H)

Tripeptide

MeO-N-Me-D-Val-Leu-Leu-NH2

Tripeptide MeO-N-Me-D-Val-Leu-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (278mg, 100% yield).

Pentapeptide

MeO-D-Phe-Leu-D-MeVal-Leu-Leu-NHBoc

Pentapeptide MeO-D-Phe-Leu-N-Me-D-Val-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 278 mg (0.63 mmols, 1.1 equivalents) of amine MeO-N-Me-D-Val-Leu-Leu-NH2, 217 mg (0.57 mmols, 1.0 equivalents) of acid HO-D-Phe-Leu-NHBoc, 0.40 mL (5 equivalents) of DIPEA, 172 mg (0.57 mmols, 1.0 equivalents) of DEPBT, and 45 mg (0.11 mmols, 0.2 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (229mg, 55% yield).

Rf: 0.35 (EtOAc: Hex 1:1

1H NMR (400 MHz, CD3OD): δ 0.7–0.9 (m, 24H), 1.4 (s, 9H), 1.5–1.7 (m, 6H), 3.1 (s, 3H), 3.2–3.4 (m, 2H), 3.5 (s, 3H), 4.1 (d, αH), 4.2 (d, αH), 4.4 (t, αH), 4.5–4.6 (m, 2αH), 4.6–4.7 (m, 2H), 5.0 (d, 1H), 6.7 (d, 1H), 6.8 (m, 1H), 7.0 (d, 1H), 7.1–7.3 (m, 5H)

Macrocycle

D-Phe-Leu-N-Me-D-Val-Leu-Leu

Macrocycle D-Phe-Leu-N-Me-D-Val-Leu-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 193.2 mg (0.31 mmols, 1.0 equivalents) of linear pentapeptide, 0.6 mL (11 equivalents) of DIPEA, 50.3 mg (0.16 mmols, 0.5 equivalents) of TBTU, 83.3 mg (0.22 mmols, 0.7 equivalents) HATU, and 46.8 mg (0.16 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (15mg, 8% yield).

Rf: 0.25 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 24H), 1.3–1.8 (m, 9H), 2.2 (m, 1H), 2.8 (s, 3H), 2.9–3.1 (m, 2H), 4.0 (m, αH), 4.3 (m, αH), 4.5 (m, 2αH), 4.8 (m, αH), 7.0 (d, 1H), 7.2–7.3 (m, 5H), 7.6 (d, 1H), 8.3 (m, 1H), 8.7 (s, 1H)

LCMS: m/z calcd for C33H53N5O5 (M+1) = 600.4, found 600.7

4.3.9 Synthesis of Compound 37

Dipeptide

MeO-Phe-Leu-NHBoc

Dipeptide MeO-Phe-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 476 mg (2.2 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (4 equivalents) of DIPEA, 708 mg (2.2 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (682mg, 87% yield).

Rf: 0.9 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m, 3H), 3.0–3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.2 (br, 1H), 4.8 (br, αH), 4.8–5.0 (q, αH), 6.5 (m, 1H), 7.1–7.4 (m, 5H)

Dipeptide

MeO-Phe-Leu-NH2

Dipeptide MeO-Phe-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (508mg, 100% yield).

Tripeptide

MeO-Phe-Leu-D-Val-NHBoc

Tripeptide MeO-Phe-Leu-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 960 mg (2.5 mmols, 1.1 equivalents) of amine MeO-Phe-Leu-NH2, 485 mg (2.2 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 1.6 mL (4 equivalents) of DIPEA, 788 mg (2.5 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (951 mg, 87% yield).

Rf: 0.7 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.9–1.1 (m, 12H), 1.5 (s, 9H), 1.6–1.7 (m, 2H), 1.8 (s, 1H), 2.1 (m, 1H), 3.1 (m, 2H), 3.7 (s 3H), 3.9 (dd, 1H), 4.4 (br, αH), 4.8 (dd, αH), 5.0 (d, αH), 6.3 (s, 1H), 6.6 (d, 1H), 7.1–7.3 (m, 5H)

Tripeptide

MeO-Phe-Leu-D-Val-NH2

Tripeptide MeO-Phe-Leu-D-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (758mg, 100% yield).

Dipeptide

MeO-D-Leu-Leu-NHBoc

Dipeptide MeO-D-Leu-Leu-NHBoc was synthesized following the7 “General peptide Synthesis” procedure. Utilizing 801 mg (4.4 mmols, 1.1 equivalents) of amine MeO-D-Leu-NH2, 1.0 g (4.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 2.8 mL (4 equivalents) of DIPEA, 1.5 g (4.8 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (1.4 g, 99% yield).

Rf: 0.9 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.6–1.8 (t, 6H), 3.7 (s, 3H), 4.0–4.1 (dd, αH), 4.5–4.7 (m, αH), 4.8–4.9 (br, 1H), 6.4 (d, 1H)

Dipeptide

HO-D-Leu-Leu-NHBoc

Dipeptide HO-D-Leu-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (1.2 g, 82% yield).

Pentapeptide

MeO-Phe-Leu-D-Val-D-Leu-Leu-NHBoc

Pentapeptide MeO-Phe-Leu-D-Val-D-Leu-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 758 mg (1.9 mmols, 1.0 equivalents) of amine MeO-Phe-Leu-D-Val-NH2, 667 mg (1.9 mmols, 1.0 equivalents) of acid HO-D-Leu-Leu-NHBoc, 3.6 mL (11 equivalents) of DIPEA, 373 mg (1.2 mmol, 0.6 equivalents) of TBTU, 116 mg (0.38 mmols, 0.2 equivalents) of DEPBT, and 441 mg (1.1 mmols, 0.6 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (379mg, 27% yield).

Rf: 0.4 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 24H), 1.4 (s, 9H), 1.5–1.7 (m, 6H), 2.8 (s, 3H), 3.0–3.2 (m, 1H), 3.3 (d, 1H), 3.6 (s, 3H), 4.1 (d, αH), 4.2 (d, αH), 4.4 (t, αH), 4.5–4.6 (m, 2αH), 4.6–4.7 (m, 2H), 6.1 (d, 1H), 7.1–7.3 (m, 5H), 7.4 (m, 2H), 7.6 (d, 1H),

Macrocycle

Phe-Leu-D-Val-D-Leu-Leu

Macrocycle Phe-Leu-D-Val-D-Leu-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 257 mg (0.43 mmols, 1.0 equivalents) of linear pentapeptide, 0.9 mL (12 equivalents) of DIPEA, 68.3 mg (0.21 mmols, 0.5 equivalents) of TBTU, 113 mg (0.30 mmols, 0.7 equivalents) HATU, and 63.7 mg (0.21 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (1.4mg, 1% yield).

Rf: 0.25 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.7–1.0 (m, 24H), 1.3–1.8 (m, 9H), 2.0 (m, 1H), 2.9–3.1 (m, 2H), 3.6 (m, αH), 3.8 (m, αH), 4.2 (m, αH), 4.5 (m, αH), 4.6 (m, αH), 7.1–7.3 (m, 5H), 7.2 (d, 1H), 7.6 (d, 1H), 8.2 (d, 1H), 8.6 (d, 1H), 8.7 (s, 1H)

LCMS: m/z calcd for C32H51N5O5 (M+1) = 586.4, found 587.5

4.3.10 Synthesis of Compound 49

Dipeptide

MeO-Phe-D-MePhe-NBoc

Dipeptide MeO-Phe-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 515.5 mg (2.4 mmols, 1.1 equiv.) of amine OMe-Phe-NH2, 606 mg (2.2.0 mmols, 1.0 equiv.) of acid HO-N-Me-D-Phe-NBoc, 1.52 mL (4 equiv.) of DIPEA, 767 mg (2.4 mmols, 1.1 equiv.) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (436.7mg, 55% yield).

Rf: 0.5 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 1.4 (s, 9H), 1.6 (m, 2H), 2.6–2.8 (d, 3H), 3.2–3.2 (m, 4H), 3.7 (s, 3H), 4.6–5.0 (m, 2αH), 6.9–7.1 (s, 1H), 7.2–7.4 (m, 10H), 8.1 (m, 1H)

Dipeptide

MeO-Phe-N-Me-D-Phe-NH

Dipeptide MeO-Phe-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (337.1mg, 100% yield).

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NHBoc

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 337 mg (1.0 mmols, 1.1 equiv.) of amine MeO-Phe-N-Me-D-Phe-NH, 196 mg (0.9 mmols, 1.0 equiv.) of acid HO-Val-NHBoc, 0.7 mL (4 equiv.) of DIPEA, 116 mg (0.36 mmols, 0.4 equiv.) of TBTU, and 240 mg (0.63mmols, 0.7 equiv.) The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (324mg, 63% yield).

Rf: 0.5 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.5–0.7 (m, 6H), 1.4 (s, 9H), 1.5 (m, 1H), 2.9 (s, 3H), 3.0–3.4 (m, 4H), 3.7 (s, 3H), 4.2 (m, αH), 4.8 (m, αH), 5.1 (d, 1H), 5.4 (m, αH), 6.8 (d, 1H), 7.0–7.4 (m, 10H)

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NH2

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (264mg, 100% yield).

Dipeptide

MeO-Cha-Leu-NHBoc

Dipeptide MeO-Cha-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 489.1 mg (2.2 mmols, 1.1 equiva.) of amine MeO-Cha-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (8 equiv.) of DIPEA, 708 mg (2.2 mmols, 1.0 equiv.) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (710mg, 88% yield).

Rf: 0.5 (EtOAc: Hex 1:2)

1H NMR (200 MHz, CDCI3): δ 0.8–1.0 (m, 6H), 1.1–1.3 (m, 4H), 1.4 (s, 9H), 1.6–1.8 (m, 9H), 3.7 (s, 3H), 4.0–4.2 (dd, αH), 4.5–4.7 (m, αH), 4.8–4.9 (br, 1H), 6.2 (d, 1H)

Dipeptide

HO-Cha-Leu-NHBoc

Dipeptide HO-Cha-Leu-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (674mg, 99% yield).

Pentapeptide

MeO-Phe-N-Me-D-Phe-Val-Cha-Leu-NHBoc

Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Cha-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 264 mg (0.6 mmols, 1.1 equiv.) of amine MeO-Phe-N-Me-D-Phe-Val-NH2, 217.4 mg (0.54 mmols, 1.0 equiv.) of acid HO-Cha-Leu-NHBoc, 0.4 mL (4 equiv.) of DIPEA, 70 mg (0.22 mmols, 0.4 equiv.) of TBTU, and 165 mg (0.43 mmols, 0.8 equiv.) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (210mg, 60% yield).

Rf: 0.5 (EtOAc: Hex 2:1)

1H NMR (400 MHz, CD3OD): δ 0.4–0.6 (dd, 3H), 0.6–0.8 (dd, 3H), 0.8–1.0 (m, 6H), 1.0–1.2 (m, 4H), 1.4 (s, 9H), 1.6–1.8 (m, 9H), 3.0 (s, 3H), 2.8–3.1 (m, 2H), 3.1–3.4 (m, 2H), 3.7 (s, 3H), 4.1 (m, αH), 4.3–4.4 (m, αH), 4.4–4.6 (m, αH), 4.7–4.9 (m, αH), 5.0 (br, 1H), 5.4–5.5 (m, αH), 6.4 (d, 1H), 6.6 (d, 1H), 6.8 (d, 1H), 7.0–7.4 (m, 10H)

Macrocycle

Phe-N-Me-D-Phe-Val-Cha-Leu

Macrocycle Phe-N-Me-D-Phe-Val-Cha-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 210 mg (0.25 mmols, 1.0 equiv.) of linear pentapeptide, 0.2 mL (4 equiv.) of DIPEA, 57 mg (0.18 mmols, 0.7 equivalents) of TBTU, 67 mg (0.18 mmols, 0.7 equivalents) HATU, and 53 mg (0.18 mmols, 0.7 equiv.) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (28mg, 14% yield).

Rf: 0.5 (EtOAc: Hex 3:1)

1H NMR (400 MHz, CD3OD): δ 0.4–0.5 (dd, 3H), 0.6–0.7 (dd, 3H), 0.8–1.0 (m, 6H), 1.0–1.2 (m, 4H), 1.6–1.8 (m, 9H), 3.0 (s, 3H), 2.8–3.1 (m, 4H), 4.1 (m, αH), 4.2–4.3 (m, αH), 4.4–4.7 (m, 2αH), 5.1–5.2 (m, αH), 7.0–7.4 (m, 10H)

LCMS: m/z calcd for C33H51N5O5 (M+1) = 598.39, found 598.3

4.3.11 Synthesis of Compound 60

Dipeptide

MeO-Phe-N-Me-D-Phe-NBoc

Dipeptide MeO-Phe-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 258 mg (1.20 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 500 mg (1.09 mmols, 1.0 equivalents) of acid HO-N-Me-D-Phe-NBoc, 0.95 mL (5 equivalents) of DIPEA, and 385 mg (1.20 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (480mg, 99% yield).

Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 9H), 2.7 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.7 (m, 2αH), 7.1–7.3 (dd, 10H).

Dipeptide

MeO-Phe-N-Me-D-Phe-NH

Dipeptide MeO-Phe-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (371mg, 100% yield).

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NHBoc

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 371 mg (1.09 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-NH, 215mg (0.99 mmols, 1.0 equivalents) of acid HO-Val-NHBoc, 0.95 mL (5 equivalents) of DIPEA, 349 mg (1.09 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (491mg, 92% yield). Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.0 (d, 6H), 1.4 (s, 9H), 2.9 (s, 3H), 3.1 (t, 4H), 3.7 (s, 3H), 4.1–4.3 (m, 2αH), 4.7–4.8 (m, 2αH), 5.0–5.1 (br, 1H), 5.5 (m, 1H), 7.2–7.3 (dd, 10H)

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NH2

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (400mg, 100% yield).

Dipeptide

MeO-Cha-Ser(Bn)-NHBoc

Dipeptide MeO-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 412 mg (1.86 mmols, 1.1 equivalents) of amine MeO-Cha-NH2, 500 mg (1.69 mmols, 1.0 equivalents) of acid HO-Ser(Bn)-NHBoc, 1.4 mL (5 equivalents) of DIPEA, 597 mg (1.86 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (781mg, 99% yield). Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 20H), 1.6–1.7 (m, 2H), 3.7 (s, 3H), 3.8 (d, 2H), 4.1 (m, 2H), 4.4 (s, 2H), 4.5 (d, 2αH), 7.3–7.4 (dd, 5H)

Dipeptide

HO-Cha-Ser(Bn)-NHBoc

Dipeptide HO-Cha-Ser(Bn)-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (715mg, 94% yield).

Pentapeptide

MeO-Phe-N-Me-D-Phe-Val-Cha-Ser(Bn)-NHBoc

Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 400 mg (0.91 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-Val-NH2, 370 mg (0.82 mmols, 1.0 equivalents) of acid HO-Cha-Ser(Bn)-NHBoc, 0.57 mL (4 equivalents) of DIPEA, 132 mg (0.41 mmols, 0.5 equivalents) of TBTU, and 156 mg (0.41 mmols, 0.5 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (279mg, 39% yield). Rf: 0.5 (EtOAc: Hex 1:1); 1H NMR (500 MHz, CDCI3): δ 1.1–1.2 (br, 6H), 1.4 (s, 20H), 2.0 (s, 3H), 3.0–3.1 (m, 2H), 3.1–3.2 (m, 2H), 3.4 (dd, 1H), 3.6 (m, 1H), 3.7 (s, 3H), 3.9 (m, 2H), 4.3 (br, αH), 4.4 (m, αH), 4.5 (m, αH), 4.5 (s, 2H), 4.6 (m, αH), 4.7–4.8 (m, αH), 5.5 (m, 2H), 6.7 (m, 1H), 7.0 (d, 1H), 7.2–7.4 (m, 15H)

Macrocycle

Phe-D-MePhe-Val-Cha-Ser(Bn)

Macrocycle Phe-D-MePhe-Val-Cha-Ser(Bn) was synthesized following the “Macrocyclization procedure”. Utilizing 242 mg (0.32 mmols, 1.0 equivalents) of linear pentapeptide, 0.22 mL (4 equivalents) of DIPEA, 51 mg (0.16 mmols, 0.5 equivalents) of TBTU, 61 mg (0.16 mmols, 0.5 equivalents) HATU, and 48 mg (0.16 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (7.2mg, 3.5% yield). Rf: 0.5 (EtOAc: Hex 9:1); 1H NMR (400 MHz, CD3OD): 0.9–1.0 (br, 6H), 1.5–1.7 (m, 11H), 1.9 (m, 2H), 3.0–3.1 (m, 4H), 3.6–3.7 (m, 2H), 4.1 (m, αH), 4.2 (m, αH), 4.3 (m, 2αH), 4.4 (m, αH), 4.5 (m, 1H), 5.1 (m, 1H), 5.3 (m, 1H), 5.4 (m, 1H), 7.1–7.3 (dd, 10H); LCMS: m/z calcd for C39H49N5O6 (M+1) = 648, found 650.2

4.3.12 Synthesis of Compound 61

Macrocycle

D-Phe-Leu-N-Me-Val-D-Leu-D-Phe

Macrocycle D-Phe-Leu-N-Me-Val-D-Leu-D-Phe was synthesized following the “Macrocyclization procedure”. Utilizing 151 mg (0.23 mmols, 1.0 equivalents) of linear pentapeptide, 0.61 mL (8 equivalents) of DIPEA, 45.8 mg (0.14 mmols, .6 equivalents) of TBTU, 52.5 mg (0.13 mmols, .6 equivalents) HATU, and 41 mg (0.14 mmols, .6 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (42mg, 28% yield).

Rf: 0.5 (EtOAc: Hex 4:1)

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 18H), 1.3 (s, 1H), 1.5 (t, 4H), 1.7 (m, 1H), 2.8 (m, 1H), 3.0 (d, 2H), 3.2 (m, 2H), 3.2(s, 3H), 4.2 (m, 3αH), 4.5 (m, 2αH), 7.1–7.3 (m, 10H), 7.7 (s, 1H), 7.9 (s, 1H), 8.4 (d, 1H), 8.5 (s, 1H)

LCMS: m/z calcd for C36H51N5O5 (M+1) = 633.82, found 634.7

4.3.13 Synthesis of Compound 62

Macrocycle

Phe-Leu-N-Me-Val-Leu-D-Phe

Macrocycle Phe-Leu-N-Me-Val-Leu-D-Phe was synthesized following the “Macrocyclization procedure”. Utilizing 287 mg (0.44 mmols, 1.0 equivalents) of linear pentapeptide, 0.61 mL (8 equivalents) of DIPEA, 84 mg (0.26 mmols, .6 equivalents) of TBTU, 100 mg (0.26 mmols, .6 equivalents) HATU, and 78 mg (0.26 mmols, .6 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (75mg, 26% yield).

Rf: 0.5 (EtOAc: Hex 4:1)

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 18H), 1.3–1.8 (m, 6H), 2.7 (m, 1H), 2.9 (m, 2H), 3.1 (m, 2H), 3.2 (s, 3H), 4.4 (m, 3αH), 4.7(m, 2αH), 7.1–7.3 (m, 10H), 7.7 (s, 1H), 8.0 (s, 1H), 8.2 (d, 1H), 8.7 (s, 1H)

LCMS: m/z calcd for C36H51N5O5 (M+1) = 633.82, found 635.1

4.3.14 Synthesis of Compound 63

Dipeptide

MeO-Phe-Leu-NHBoc

Dipeptide MeO-Phe-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 951 mg (4.4 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 1000 mg (4.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 2.8 mL (4 equivalents) of DIPEA, 1546 mg (4.8 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (1564mg, 99% yield).

Rf: 0.8 (EtOAc: Hex 1:1)

1H NMR (200 MHz, CDCI3): δ 0.8–1.0 (m, 6H), 1.4 (s, 9H), 1.6 (m, 2H), 3.0–3.1 (m, 2H), 3.7 (s, 3H), 4.8–5.1 (s, 2H), 4.1 (m, αH), 4,8 (m, αH), 6.5 (d, 1H), 7.0–7.3 (m, 5H)

Dipeptide

MeO-Phe-Leu-NH2

Dipeptide MeO-Phe-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (1165mg, 100% yield).

Tripeptide

MeO-Phe-Leu-N-Me-Val-NBoc

Tripeptide MeO-Phe-Leu-N-Me-Val-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 510 mg (1.74 mmols, 1.1 equivalents) of amine MeO-Phe-Leu-NH2, 367mg (1.58 mmols, 1.0 equivalents) of acid HO-N-Me-Val-NBoc, 1.1 mL (4 equivalents) of DIPEA, 723 mg (1.9 mmols, 1.2 equivalents) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (585 mg, 73% yield).

Rf: 0.6 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CDCI3): δ 0.9–1.1 (m, 12H), 1.5 (s, 9H), 1.6–1.8 (m, 1H), 2.3 (m, 2H), 2.8 (s, 3H), 3.1 (m, 2H), 3.7 (s, 3H), 4.0 (d, αH), 4.4 (m, αH), 4.8 (m, αH), 6.3 (d, 1H), 6.5 (d, 1H), 7.1–7.3 (m, 5H)

Tripeptide

MeO-Phe-Leu-N-Me-Val-NH

Tripeptide MeO-Phe-Leu-N-Me-Val-NH was synthesized following the “General amine deprotection”. This tripeptide was taken on to the next reaction without further purification or characterization. (468mg, 100% yield).

Tetrapeptide

MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-NHBoc

Tetrapeptide MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 468 mg (1.1 mmols, 1.1 equivalents) of amine MeO-Phe-Leu-N-Me-Val-NH, 436 mg (1.0 mmols, 1.0 equivalents) of acid HO-Lys(2-CI-Cbz)-NHBoc, 1.27 mL (7 equivalents) of DIPEA, 135 mg (0.4 mmols, 0.4 equivalents) of TBTU, and 320 mg (0.8 mmols, 0.8 equivalents) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (675mg, 80% yield).

Rf: 0.35 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.4(s, 9H), 1.4–1.8 (m, 9H), 2.3 (m, 2H), 3.0 (s, 3H), 3.1–3.2 (m, 2H), 3.7 (s, 3H), 4.3 (m, αH), 4.4 (d, 1H), 4.6 (m, αH), 4.8 (m, αH), 4.9 (m, αH), 5.2 (s, 2H), 5.3 (d, 1H), 6.4 (d, 1H), 6.5 (d, 1H), 7.1–7.4 (m, 9H)

Tetrapeptide

MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-NH2

Tetrapeptide MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-NH2 was synthesized following the “General amine deprotection”. This tetrapeptide was taken on to the next reaction without further purification or characterization. (590mg, 100% yield).

Pentapeptide

MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-D-Phe-NHBoc

Pentapeptide MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-D-Phe-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 570 mg (0.8 mmols, 1.1 equivalents) of amine MeO-Phe-Leu-N-Me-Val-D-Lys(2-CI-Cbz)-NH2, 195 mg (0.7 mmols, 1.0 equivalents) of acid HO-D-Phe-NHBoc, 0.6 mL (5 equivalents) of DIPEA, 95 mg (0.3 mmols, 0.4 equivalents) of TBTU, and 224 mg (0.6 mmols, 0.8 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (549mg, 79% yield).

Rf: 0.3 (EtOAc: Hex 1:1)

1H NMR (400 MHz, CD3OD): δ 0.7–0.9 (m, 12H), 1.4 (s, 9H), 1.3–1.7 (m, 9H), 2.3 (m, 2H), 3.0 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), (d, αH), 4.3–4.4 (m, 2αH), 4.8–4.9 (m, 2αH), 5.0 (br, αH), 5.2 (s, 2H), 6.4–6.5 (dd, 2H), 6.9 (d, 1H), 7.1–7.5 (m, 14H)

Macrocycle

Phe-Leu-N-Me-Val-D-Lys(2-Cl-Cbz)-D-Phe

Macrocycle Phe-Leu-N-Me-Val-D-Lys(2-Cl-Cbz)-D-Phe was synthesized following the “Macrocyclization procedure”. Utilizing 228 mg (0.27 mmols, 1.0 equivalents) of linear pentapeptide, 0.38 mL (8 equivalents) of DIPEA, 43.8 mg (0.14 mmols, 0.5 equivalents) of TBTU, 62.3 mg (0.16 mmols, 0.6 equivalents) HATU, and 49 mg (0.16 mmols, 0.6 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) yield the macrocycle (37.5mg, 16.8% yield).

Rf: 0.5 (EtOAc: Hex 4:1)

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 12H), 1.2–1.6 (m, 8H), 1.7–1.9 (m, 1H), 2.1–2.3 (m, 2H), 2.9 (s, 3H), 2.7– 3.1 (m, 4H), 4.1–4.3 (m, 3αH), 4.5 (m, αH), 4.7 (m, αH), 5.1 (d, 1H), 5.2 (s, 2H), 7.0–7.4 (m, 15H)

LCMS: m/z calcd for C44H57CIN6O7 (M+1) = 818.41, found 818.2

4.3.15 Removal of Carbobenzyloxy groups via Acid to yield compound 64

Macrocycle

Phe-Leu-N-Me-D-Val-D-Lys-D-Phe

Utilizing 26 mg (0.03 mmols, 1.0 equivalents) of compound 140 and 0.32 mL of HBr to remove the protecting group on the Lysine at Position D-Lys(2-CI-Cbz). The crude reaction was taken on to the next reaction without further purification or characterization (20.8mg, 100% yield).

1H NMR (400 MHz, CD3OD): δ 0.8–1.0 (m, 12H), 1.2–1.7 (m, 8H), 1.7–1.9 (m, 1H), 2.1–2.3 (m, 2H), 2.9 (s, 3H) 2.8– 2.9 (m, 4H), 4.2 (m, αH), 4.3 (m, 2αH), 4.5 (m, αH), 4.7 (m, αH), 5.0 (d, 1H), 7.0–7.3 (m, 9H)

LCMS: m/z calcd for C36H52N6O5 (M+1) = 649.84, found 649.6

4.3.16 Synthesis of Compound 65

Dipeptide

MeO-Phe-N-Me-D-Phe-NBoc

Dipeptide MeO-Phe-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 258 mg (1.20 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 500 mg (1.09 mmols, 1.0 equivalents) of acid HO-N-Me-D-Phe-NBoc, 0.95 mL (5 equivalents) of DIPEA, and 385 mg (1.20 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (480mg, 99% yield).

Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 9H), 2.7 (s, 3H), 3.1–3.2 (m, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.7 (m, 2αH), 7.1–7.3 (dd, 10H)

Dipeptide

MeO-Phe-N-Me-D-Phe-NH

Dipeptide MeO-Phe-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (371mg, 100% yield).

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NHBoc

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 371 mg (1.09 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-NH, 215mg (0.99 mmols, 1.0 equivalents) of acid HO-Val-NHBoc, 0.95 mL (5 equivalents) of DIPEA, 349 mg (1.09 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (491mg, 92% yield).

Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.0 (d, 6H), 1.4 (s, 9H), 2.9 (s, 3H), 3.1 (t, 4H), 3.7 (s, 3H), 4.1–4.3 (m, 2αH), 4.7–4.8 (m, 2αH), 5.0–5.1 (br, 1H), 5.5 (m, 1H), 7.2–7.3 (dd, 10H)

Tripeptide

MeO-Phe-N-Me-D-Phe-Val-NH2

Tripeptide MeO-Phe-N-Me-D-Phe-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (400mg, 100% yield).

Dipeptide

MeO-Cha-Ser(Bn)-NHBoc

Dipeptide MeO-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 412 mg (1.86 mmols, 1.1 equivalents) of amine MeO-Cha-NH2, 500 mg (1.69 mmols, 1.0 equivalents) of acid HO-Ser(Bn)-NHBoc, 1.4 mL (5 equivalents) of DIPEA, 597 mg (1.86 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (781mg, 99% yield). Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 20H), 1.6–1.7 (m, 2H), 3.7 (s, 3H), 3.8 (d, 2H), 4.1 (m, 2H), 4.4 (s, 2H), 4.5 (d, 2αH), 7.3–7.4 (dd, 5H).

Dipeptide

HO-Cha-Ser(Bn)-NHBoc

Dipeptide HO-Cha-Ser(Bn)-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (715mg, 94% yield).

Pentapeptide

MeO-Phe-N-Me-D-Phe-Val-Cha-Ser(Bn)-NHBoc

Pentapeptide MeO-Phe-N-Me-D-Phe-Val-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 400 mg (0.91 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-NH, 370 mg (0.82 mmols, 1.0 equivalents) of acid HO-Cha-Ser(Bn)-NHBoc, 0.57 mL (4 equivalents) of DIPEA, 132 mg (0.41 mmols, 0.5 equivalents) of TBTU, and 156 mg (0.41 mmols, 0.5 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (279mg, 39% yield). Rf: 0.5 (EtOAc: Hex 1:1); 1H NMR (500 MHz, CDCI3): δ 1.1–1.2 (br, 6H), 1.4 (s, 20H), 2.0 (s, 3H), 3.0–3.1 (m, 2H), 3.1–3.2 (m, 2H), 3.4 (dd, 1H), 3.6 (m, 1H), 3.7 (s, 3H), 3.9 (m, 2H), 4.3 (br, αH), 4.4 (m, αH), 4.5 (m, αH), 4.5 (s, 2H), 4.6 (m, αH), 4.7–4.8 (m, αH), 5.5 (m, 2H), 6.7 (m, 1H), 7.0 (d, 1H), 7.2–7.4 (m, 15H).

Macrocycle

Phe-N-Me-D-Phe-Val-Cha-Ser(Bn)

Macrocycle Phe-N-Me-D-Phe-Val-Cha-Ser(Bn) was synthesized following the “Macrocyclization procedure”. Utilizing 242 mg (0.32 mmols, 1.0 equivalents) of linear pentapeptide, 0.22 mL (4 equivalents) of DIPEA, 51 mg (0.16 mmols, 0.5 equivalents) of TBTU, 61 mg (0.16 mmols, 0.5 equivalents) HATU, and 48 mg (0.16 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (7.2mg, 2.0% yield). Rf: 0.5 (EtOAc: Hex 9:1); 1H NMR (400 MHz, CD3OD): δ 0.9–1.0 (br, 6H), 1.4–1.6 (m, 11H), 1.6–1.7 (m, 2H), 2.2 (br, 1H), 3.0–3.1 (m, 4H), 3.6–3.8 (m, 2H), 4.4–4.5 (m, 2αH), 4.5–4.6 (br, 2αH), 5.1 (m, 2H), 7.1 (m, 2H), 7.1–7.3 (dd, 15H)LCMS: m/z calcd for C43H55N5O6 (M+1) = 738, found 738.36.

4.3.17 Synthesis of Compound 66

Dipeptide

MeO-Phe-N-Me-D-Phe-NBoc

Dipeptide MeO-Phe-N-Me-D-Phe-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 258 mg (1.20 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 500 mg (1.09 mmols, 1.0 equivalents) of acid HO-N-Me-D-Phe-NBoc, 1.0 mL (5 equivalents) of DIPEA, and 385 mg (1.20 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (480mg, 99% yield).

Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 9H), 2.7–2.8 (s, 3H), 3.1–3.2 (s, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 1H), 4.9–5.0 (br, 2αH), 7.2–7.4 (dd, 10H)

Dipeptide

MeO-Phe-N-Me-D-Phe-NH

Dipeptide MeO-Phe-N-Me-D-Phe-NH was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (371mg, 100% yield).

Tripeptide

MeO-Phe-N-Me-D-Phe-D-Val-NHBoc

Tripeptide MeO-Phe-N-Me-D-Phe-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 371 mg (1.09 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-NH, 215mg (0.99 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 0.95 mL (5 equivalents) of DIPEA, 349 mg (1.09 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (512mg, 96% yield). Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.0–1.1 (m, 6H), 1.4 (s, 9H), 2.9 (m, 1H), 3.0 (s, 3H), 3.4–3.5 (br, 4H), 3.7 (s, 3H), 4.1–4.2 (m, 2αH), 4.8 (m, αH), 5.0 (br, 1H), 5.4 (br, 1H), 7.2–7.3 (m, 10H).

Tripeptide

MeO-Phe-N-Me-D-Phe-D-Val-NH2

Tripeptide MeO-Phe-N-Me-D-Phe-D-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (417mg, 100% yield).

Dipeptide

MeO-Cha-Ser(Bn)-NHBoc

Dipeptide MeO-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 412 mg (1.86 mmols, 1.1 equivalents) of amine MeO-Cha-NH2, 500 mg (1.69 mmols, 1.0 equivalents) of acid HO-Ser(Bn)-NHBoc, 1.4 mL (5 equivalents) of DIPEA, 597 mg (1.86 mmols, 1.1 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (780mg, 99% yield). Rf: 0.5 (EtOAc: Hex 2:3); 1H NMR (200 MHz, CDCI3): δ 1.4 (s, 20H), 1.5–1.6 (m, 2H), 3.7 (s, 3H), 3.9 (dd, 2H), 4.1 (br, 2H), 4.5 (s, 2H), 4.6 (m, 2αH), 7.2–7.3 (s, 5H)

Dipeptide

HO-Cha-Ser(Bn)-NHBoc

Dipeptide HO-Cha-Ser(Bn)-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (766mg, 99% yield).

Pentapeptide

MeO-Phe-N-Me-D-Phe-D-Val-Cha-Ser(Bn)-NHBoc

Pentapeptide MeO-Phe-N-Me-D-Phe-D-Val-Cha-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 417 mg (0.94 mmols, 1.1 equivalents) of amine MeO-Phe-N-Me-D-Phe-D-Val-NH2, 386 mg (0.86 mmols, 1.0 equivalents) of acid HO-Cha-Ser(Bn)-NHBoc, 0.60 mL (4 equivalents) of DIPEA, 138 mg (0.43 mmols, 0.5 equivalents) of TBTU, and 164 mg (0.43 mmols, 0.5 equivalents) HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (210mg, 28% yield). Rf: 0.5 (EtOAc: Hex 3:2); 1H NMR (500 MHz, CDCI3): δ 0.9–1.0 (m, 6H), 1.4 (s, 9H), 1.5–1.6 (s, 11H), 1.8 (m, 2H), 2.8 (s, 3H), 3.0 (m, 2H), 3.2 (m, 2H), 3.6 (s, 3H), 3.8 (m, 1H), 4.2 (m, 2αH), 4.4 (m, 2αH), 4.5 (m, 2H), 4.7 (m, αH), 5.0 (br, 1H), 6.2 (d, 1H), 6.4 (d, 1H), 6.5 (d, 1H), 7.1–7.3 (dd, 15H)

Macrocycle

Phe-N-Me-D-Phe-D-Val-Cha-Ser(Bn)

Macrocycle Phe-N-Me-D-Phe-D-Val-Cha-Ser(Bn) was synthesized following the “Macrocyclization procedure”. Utilizing 182 mg (0.24 mmols, 1.0 equivalents) of linear pentapeptide, 0.16 mL (4 equivalents) of DIPEA, 39 mg (0.12 mmols, 0.5 equivalents) of TBTU, 46 mg (0.12 mmols, 0.5 equivalents) HATU, and 36 mg (0.12 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by reverse phase-HPLC to yield the macrocycle (1.0mg, 1.0% yield). Rf: 0.5 (EtOAc: Hex 3:1); 1H NMR (400 MHz, CD3OD): δ 0.9–1.0 (dd, 6H), 1.2–1.3 (m, 4H), 1.3–1.4 (m, 6H), 1.7–1.8 (m, 1H), 1.9 (br, 2H), 2.9 (s, 3H), 3.0–3.1 (m, 3H), 3.1–3.2 (m, 3H), 3.7 (m, 2H), 3.8 (m, 2H), 3.9 (m, αH), 4.2 (m, 2αH), 4.4 (m, 2αH, 4.5 (s, 2H), 7.1–7.3 (dd, 15H); LCMS: m/z calcd for C43H55N5O6 (M+1) = 738, found 738.5

4.3.18 Synthesis of Compound 67

Dipeptide

MeO-D-Phe-D-Val-NHBoc

Dipeptide MeO-D-Phe-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 476 mg (2.2 mmols, 1.1 equivalents) of amine MeO-D-Phe-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 1.4 mL (4 equivalents) of DIPEA, and 773 mg (2.4 mmols, 1.2 equivalents) of TBTU. The crude reaction was washed 2× 100ml pH=1 water and 10× 100ml saturated NaHCO3 to yield the dipeptide (788mg, 95% yield). Rf: 0.5 (EtOAc: Hex 35:65); 1H NMR (200 MHz, CDCI3): δ 0.95 (d, 6H), 1.5 (s, 9H), 1.6–1.8 (m, 3H), 3.2 (m, 2H), 3.7 (s, 3H), 4.0–4.1 (m, αH), 4.8–5.0 (m, αH), 6.4–6.6 (d, 1H), 7.1–7.4 (m, 5H).

Dipeptide

MeO-D-Phe-D-Val-NH2

Dipeptide MeO-D-Phe-D-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (596mg, 100% yield).

Tripeptide

MeO-D-Phe-D-Leu-D-Val-NHBoc

Tripeptide MeO-D-Phe-D-Leu-D-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 596 mg (1.9 mmols, 1.1 equivalents) of amine MeO-D-Phe-D-Leu-NH2, 379mg (1.7 mmols, 1.0 equivalents) of acid HO-D-Val-NHBoc, 1.2 mL (5 equivalents) of DIPEA, 672 mg (0.85 mmols, 2.0 equivalents) of TBTU. The crude reaction was washed 3× 100ml saturated NaCl. It was then purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (857mg, 94% yield). Rf: 0.5 (EtOAc: Hex 1:1); 1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 12H), 1.5 (s, 12H), 1.6–1.8 (m, 3H), 2.1 (m, 1H), 3.1 (d, 2H), 3.7 (s, 3H), 3.9– 4.0 (dd, 1H), 4.4 (m, αH), 4.9 (m, αH), 5.0 (d, αH), 6.3 (d, 1H), 6.5 (d, 1H), 7.0–7.4 (m, 5H).

Tripeptide

MeO-D-Phe-D-Leu-D-Val-NH2

Tripeptide MeO-D-Phe-D-Leu-D-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (502mg, 100% yield).

Dipeptide

MeO-Leu-Ser(Bn)-NHBoc

Dipeptide MeO-Leu-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 339 mg (1.9 mmols, 1.1 equivalents) of amine MeO-Leu-NH2, 500 mg (1.7 mmols, 1.0 equivalents) of acid HO-Ser(Bn)-NHBoc, 1.2 mL (4 equivalents) of DIPEA, 652 mg (2.0 mmols, 1.2 equivalents) of TBTU. The crude reaction was washed 2× 100ml with pH 1 water and 10× 100ml with saturated NaHCO3 to yield the dipeptide (636mg, 89% yield). Rf: 0.5 (EtOAc: Hex 35:65); 1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (m, 6H), 1.4(s, 9H), 1.6–1.8 (m, 5H), 3.5 (dd, 2H), 3.7 (s, 3H), 3.9–4.0 (dd, 1H), 4.2 (m, αH), 4.5 (s, 2H), 4.6 (m, αH), 5.3 (d, 1H), 6.9 (d, 1p), 7.3 (m, 5H)

Dipeptide

HO-Leu-Ser(Bn)-NHBoc

Dipeptide HO-Leu-Ser(Bn)-NHBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (631mg, 98% yield).

Pentapeptide

MeO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NHBoc

Pentapeptide MeO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 488 mg (1.2 mmols, 1.1 equivalents) of amine MeO-D-Phe-D-Leu-D-Val-NH2, 463 mg (1.1 mmols, 1.0 equivalents) of acid HO-Leu-Ser(Bn)-NHBoc, 1.2 mL (6 equivalents) of DIPEA, and 436 mg (1.4 mmols, 1.2 equivalents) of TBTU. The crude reaction was washed 2× 100ml with pH 1 water and 10× 100ml with saturated NaHCO3 It was then purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (435mg, 52% yield). Rf: 0.5 (EtOAc: Hex 65:35); 1H NMR (400 MHz, CD3OD): δ 0.8–0.9 (m, 18H), 1.3 (s, 1H), 1.4 (s, 9H), 1.6–1.8 (m, 7H), 2.1 (m, 1H), 3.0–3.2 (m, 2H), 3.6–3.8 (m, 4H), 4.2 (m, αH), 4.3 (m, αH), 4.4 (m, 2αH), 4.5–4.7 (d, 3H), 7.1–7.3 (m, 10H), 7.8 (d, 1H), 8.0 (d, 1H), 8.1 (d, 1H)

Pentapeptide

HO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NHBoc

Pentapeptide HO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NHBoc was synthesized following the “General acid deprotection”. This pentapeptide was taken on to the next reaction without further purification or characterization. (252mg, 60% yield).

Pentapeptide

HO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NH2

Pentapeptide HO-D-Phe-D-Leu-D-Val-Leu-Ser(Bn)-NH2 was synthesized following the “General amine deprotection”. This pentapeptide was taken on to the next reaction without further purification. (191mg, 100% yield). LCMS: m/z calcd for C36H53N5O7 (M+1) =668.8, found 669.0.

Macrocycle

D-Phe-D-Leu-D-Val-Leu-Ser(Bn)

Macrocycle D-Phe-D-Leu-D-Val-Leu-Ser(Bn) was synthesized following the “Macrocyclization procedure”. Utilizing 220 mg (0.32 mmols, 1.0 equivalents) of linear pentapeptide, 0.34 mL (8 equivalents) of DIPEA, 53.0 mg (0.17 mmols, 0.5 equivalents) of TBTU, 62.7 mg (0.17 mmols, 0.5 equivalents) HATU, and 49.4 mg (0.17 mmols, 0.5 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the macrocycle (36.5mg, 17% yield). Rf: 0.5 (EtOAc: Hex 70:30); 1H NMR (500 MHz, CD3OD): δ 0.8–1.1 (m, 18H), 1.4 (s, 3H), 1.6–1.8 (m, 7H), 2.1 (d, 1H), 2.2 (m, 1H), 3.2–3.4 (m, 1H), 3.8 (m, 2H), 4.2 (m, 3αH), 4.5 (m, αH), 4.7 (m, 2αH), 6.9 (d, 1H), 7.2–7.5 (m, 10H), 7.9 (d, 1H), 8.2 (d, 1H), 8.4 (d, 1H), 8.7 (d, 1H); LCMS: m/z calcd for C36H51N5O6 (M+1) = 650.8, found 651.4.

4.3.19 Synthesis of Compound 68

Dipeptide

MeO-Phe-Leu-NHBoc

Dipeptide MeO-Phe-Leu-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 477 mg (2.2 mmols, 1.1 equivalents) of amine MeO-Phe-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-Leu-NHBoc, 1.4 mL (4 equivalents) of DIPEA, 773 mg (2.4 mmols, 1.2 equivalents) of TBTU. The crude reaction was purified by colum n chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (785mg, 99% yield).

Rf: 0.5 (EtOAc: Hex 1:4)

1H NMR (200 MHz, CDCI3): δ 0.9 (m, 6H), 1.4–1.5 (s, 9H), 1.6–1.7 (br, 3H), 3.1–3.2 (t, 2H), 3.7–3.8 (s, 3H), 4.0–4.2 (m, αH), 4.7–4.9 (m, αH, 1H), 6.5 (d, 1H), 7.0–7.2 (br, 2H), 7.3– 7.4 (s, 3H)

Dipeptide

MeO-Phe-Leu-NH2

Dipeptide MeO-Phe-Leu-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (584mg, 100% yield).

Tripeptide

MeO-Phe-Leu-Val-NHBoc

Tripeptide MeO-Phe-Leu-Val-NHBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 578 mg (1.98 mmols, 1.0 equivalents) of amine MeO-Phe-Leu-NH2, 433mg (1.94 mmols, 1.02 equivalents) of acid HO-Val-NHBoc, 1.90 mL (6 equivalents) of DIPEA, 466 mg (1.44 mmols, 0.7 equivalents) of TBTU, 275 mg (0.72 mmols, 0.4 equivalents) of HATU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the tripeptide (886 mg, 91% yield).

Rf: 0.5 (EtOAc: Hex 2:3)

1H NMR (200 MHz, CDCI3): δ 0.8–1.0 (m, 12H), 1.4–1.5 (s, 9H), 1.6–1.7 (m, 3H), 2.0–2.2 (m, 1H), 3.1 (d, 2H), 3.7 (s, 3H), 3.8–3.9 (q, αH), 4.4–4.5 (m, αH), 4.8–4.9 (q, αH), 5.0 (d, 1H), 6.3 (d, 1H), 6.4–6.5 (d, 1H), 7.1 (m, 2H), 7.2–7.4 (m, 3H)

Tripeptide

MeO-Phe-Leu-Val-NH2

Tripeptide MeO-Phe-Leu-Val-NH2 was synthesized following the “General amine deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (705mg, 100% yield).

Dipeptide

MeO-Lys(Cbz)-N-Me-Leu-NBoc

Dipeptide MeO-Lys(Cbz)-N-Me-Leu-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 742 mg (2.2 mmols, 1.1 equivalents) of amine MeO-Lys(Cbz)-NH2, 500 mg (2.0 mmols, 1.0 equivalents) of acid HO-N-Me-Leu-NBoc, 1.4 mL (4 equivalents) of DIPEA, 458 mg (1.4 mmols, 0.7 equivalents) of TBTU. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the dipeptide (1.05g, 91% yield).

Rf: 0.5 (EtOAc: Hex 2:3)

1H NMR (200 MHz, CDCI3): δ 0.9–1.0 (t, 6H), 1.2–1.4 (m, 2H), 1.5 (s, 9H), 1.6–1.7 (m, 6H), 1.8–1.9 (m, 1H), 2.7–2.8 (s, 3H), 3.1–3.2 (q, 2H), 3.7 (s, 3H), 4.5–4.7 (br, αH), 4.8–4.9 (br, αH), 5.1 (s, 2H), 6.4–6.5 (br, 1H), 6.5 (br, 1H), 7.3 (s, 1H), 7.4 (s, 4H)

Dipeptide

HO-Lys(Cbz)-N-Me-Leu-NBoc

Dipeptide HO-Lys(Cbz)-N-Me-Leu-NBoc was synthesized following the “General acid deprotection”. This dipeptide was taken on to the next reaction without further purification or characterization. (947mg, 93% yield).

Pentapeptide

MeO-Phe-Leu-Val-Lys(Cbz)-N-Me-Leu-NBoc

Pentapeptide MeO-Phe-Leu-Val-Lys(Cbz)-N-Me-Leu-NBoc was synthesized following the “General peptide Synthesis” procedure. Utilizing 705 mg (1.80 mmols, 1.1 equivalents) of amine MeO-Phe-Leu-Val-NH2, 940 mg (1.72 mmols, 1.0 equivalents) of acid HO-Lys(Cbz)-N-Me-Leu-NBoc, 2.4 mL (8 equivalents) of DIPEA, 347 mg (1.08 mmols, 0.6 equivalents) of TBTU, 342 mg (0.90 mmols, 0.50 equivalents) HATU, and 216mg (0.72 mmols, 0.4 equivalents) DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the pentapeptide (573mg, 37% yield).

Rf: 0.5 (EtOAc: Hex 7:3

1H NMR (400 MHz, CDCL3): δ 0.8–1.0 (m, 18H), 1.3–1.4 (m, 2H), 1.5 (m, 12H), 1.6–1.7 (m, 5H), 1.7–1.8 (s, 1H), 1.8–1.9 (br, 1H), 2.2 (br, 1H), 3.7–3.8 (d, 3H), 3.0–3.2 (m, 4H), 3.7 (s, 3H), 4.2 (m, αH), 4.3–4.4 (br, αH), 4.5 (br, αH), 4.6–4.7 (br, αH), 4.8 (m, αH), 4.9 (br, 1H), 5.1 (s, 2H), 6.6–6.8 (br, 2H), 6.9 (br, 1H) 7.1–7.2 (d, 2H), 7.2–7.3 (m, 5H), 7.3–7.4 (m, 4H)

Macrocycle

Phe-Leu-Val-Lys(Cbz)-N-Me-Leu

Macrocycle Phe-Leu-Val-Lys(Cbz)-N-Me-Leu was synthesized following the “Macrocyclization procedure”. Utilizing 478 mg (0.61 mmols, 1.0 equivalents) of linear pentapeptide, 0.63 mL (6 equivalents) of DIPEA, 97.3 mg (0.30 mmols, 0.50 equivalents) of TBTU, 115 mg (0.30 mmols, 0.50 equivalents) HATU, and 90.7 mg (0.30 mmols, 0.50 equivalents) of DEPBT. The crude reaction was purified by column chromatography (silica gel, EtOAc/Hex) to yield the macrocycle (47.6mg, 10.5% yield).

Rf: 0.5 (EtOAc: Hex 4:1)

1H NMR (400 MHz, CDCL3): δ 0.7–1.1 (m, 18H), 1.2 (m, 1H), 1.4–1.6 (m, 6H), 1.6–1.8 (m, 3H), 1.9 (s, 2H), 1 2.7 (s, 3H), 2.9–3.0 (m, 1H), 3.1–3.3 (m, 3H) 3.9 (t, αH), 4.3 (m, αH), 4.4 (br, αH), 4.4–4.6 (br, αH), 5.0 (m, αH), 5.1 (s, 2H, 1αH), 6.7 (br, 1H), 7.1–7.3 (m, 7H), 7.3–7.4 (m, 6H), 7.7–7.8 (s, 1H)

LCMS: m/z calcd for C41H60N6O7 (M+1) = 748.45, found 749.5

4.3.16 Removal of Carbobenzyloxy groups via Acid to yield compound 69

Utilizing 47.6 mg (0.06 mmols, 1 equivalent) of pentapeptide Phe-Leu-Val-Lys(Cbz)-N-Me-Leu, the carbobenzyloxy group was removed by dissolving the compound in 33% Hydrogen bromine (HBr) in glacial acetic acid (0.1M). The crude reaction was concentrated down in-vacuo and purified via reversed-phase HPLC. (17.2 mg, 84% yield).

Rf: 0.5 (EtOAc: Hex 5:1)

1H NMR (400 MHz, CD3OD): δ 0.7–1.1 (m, 18H), 1.2 (m, 1H), 1.4–1.6 (m, 6H), 1.6–1.8 (m, 3H), 1.9 (s, 2H), 2.7 (s, 3H), 2.9–3.0 (m, 1H), 3.1–3.3 (m, 3H), 3.9 (t, αH), 4.3 (m, αH), 4.4 (br, αH), 4.4–4.6 (br, αH), 5.0 (m, αH), 5.1 (s, 2H, 1 αH), 6.7 (br, 1H), 7.3–7.4 (m, 6H), 8.4 (s, 1H).

LCMS: m/z calcd for C33H54N6O5 (M+1) = 614.82, found 615.5

Supplementary Material

01

Supporting Information Available:

1H NMR Data and LCMS data for compounds and their intermediates are available on line and free of charge at http://www. In addition, ClogP values, % inhibition, and a list of compounds with their structural variation by position are also available in the supplementary data.

Acknowledgements

We thank San Diego State University, the Frasch foundation (658-HF07), and CSUPERB for financial support. We thank the Howell Foundation for support of R.C.V. We thank NIW T90DK07015 for support of R.C.V. We thank NIH 1U54CA132379-01A1 for support of SRM and VA.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Cueto M, Jensen PR, Fenical W. Phytochemistry. 2000;55:223. [PubMed]
2. Hwang Y, Rowley D, Rhodes D, Gertsch J, Fenical W, Bushman F. Molecular Pharmacology. 1999;55:1049. [PubMed]
3. Belofsky GN, Jensen PR, Fenical W. Tetrahedron Lett. 1999;40:2913.
4. Liu S, Gu W, D L, Ding X-Z, Ujiki M, Adrian TE, Soff GA, Silverman RB. J. Med. Chem. 2005;48:3630. [PubMed]
5. Ujiki M, Milam B, Ding X-Z, Roginsky AB, Salabat MR, Talamonti MS, Bell RH, Gu W, Silverman RB, Adrian TE. Biochemical and Biophysical Research Communications. 2006;340:1224. [PubMed]
6. Styers TJ, Kekec A, Rodriguez RA, Brown JD, Cajica J, Pan P-S, Parry E, Carroll CL, Medina I, Corral R, Lapera S, Otrubova K, Pan C-M, Mcguire KL, Mcalpine SR. Bioorganic and Medicinal Chemistry. 2006;14:5625. [PubMed]
7. Rodriguez RA, Pan P-S, Pan C-M, Ravula S, Lapera SA, Singh EK, Styers TJ, Brown JD, Cajica J, Parry E, Otrubova K, Mcalpine SR. J. Org. Chem. 2007;72:1980. [PubMed]
8. Pan PS, Curtis FA, Carroll CL, Medina I, Liotta LA, Sharples G, Mcalpine SR. Bioorg. & Med. Chem. 2006;14:4731. [PubMed]
9. Otrubova K, Styers TJ, Pan P-S, Rodriguez R, Mcguire KL, Mcalpine SR. Chem. Commun. 2006:1033. [PubMed]
10. Carroll CL, Johnston JVC, Kekec A, Brown JD, Parry E, Cajica J, Medina I, Cook KM, Corral R, Pan P-S, Mcalpine SR. Org. Lett. 2005;7:3481. [PubMed]
11. Lee Y, Silverman RB. Org. Lett. 2000;2:3743. [PubMed]
12. Gu W, Liu S, Silverman RB. Org. Lett. 2002;4:4171. [PubMed]
13. Otrubova K, Lushington GH, Vander Velde D, Mcguire KL, Mcalpine SR. J. Med. Chem. 2008;51:530. [PubMed]
14. Pan PS, Mcguire K, Mcalpine SR. Bioorg. & Med. Chem. Lett. 2007;17:5072. [PubMed]
15. Amidon GL, Lee HJ. Ann. Rev. Pharmacol. Toxicol. 1994;34:321. [PubMed]
16. Wenger RM. Prog. Allergy. 1986;38:46. [PubMed]
17. Jarvis LM. C and E News. 2006
18. Marx V. C and E News. 2005;83:17. http://pubs.acs.org/cen/business/83/i11/8311bus1.html.
19. The most recent peptide drugs include Byetta from Amylin: < http://www.drugs.com/pro/byetta.html>, and from Aileron comes stapled peptides: http://pubs.acs.org/cen/coverstory/86/8622cover.html
20. Singh EK, Sellers RP, Alexander LD, Mcalpine SR. Current opinion in Drug Discovery and Development. 2008;11:544. [PubMed]
21. Loffet A. European Peptide Society. 2002;8:1. [PubMed]
22. Murr MM, Sarr MG, Oishi AJ, Heerden JA. CA Cancer J. Clin. 1994;44:304. [PubMed]
23. Burris HA, Moore MJ, Andersen J, Greem MR, Rothenberg MI, Modiano MR, Cripps MC, Portenoy RK, Sotorniolo AM, Tarassaoff P, Nelson R, Dorr FA, Stephens CD, Vonhoff D. J. Clin. Oncol. 1997;15:2403. [PubMed]
24. Sener SF, Fremgen A, Menck HR, Winchester DP. J. Am. Coll. Surg. 1999;189:1. [PubMed]
25. Styers TJ, Rodriguez RA, Pan P-S, Mcalpine SR. Tetrahedron Lett. 2006;47:515.
26. The ClogP values were calculated using an algorithm. The logP value of a compound, which is the logarithm of its partition coefficient between n-octanol and water log(coctanol/cwater), is a well established measure of the compound’s hydrophilicity. Low hydrophilicities and therefore high logP values cause poor absorption or permeation. It has been shown for compounds to have a reasonable probability of being well absorbt their logP value must not be greater than 5.0. The distribution of calculated logP values of more than 3000 drugs on the market underlines this fact.
27. Lipinskis rules state that the ideal drug contains no more than five H-doners, no more than 10 H-acceptors, molecular weights of less than 500, and logP values ≤5.0.
28. Dipeptide and tripeptide structures were confirmed using 1H NMR. All linear pentapeptides were confirmed using LCMS and 1H NMR. (Note: 1H NMR were taken for cyclized peptides, but due to their complexity, they were not seen as the primary confirmation for cyclized compounds). See supplementary data for spectra.
29. Geistlinger TR, Mcreynolds AC, Guy RK. Chem. Biol. 2004;11:273. Multiple coupling agents reference. [PubMed]
30. Bolla ML, Azevedo EV, Smith JM, Taylor RE, Ranjit DK, Segall AM, Mcalpine SR. Org. Lett. 2003;5:109. [PubMed]
31. Two cell lines were chosen to provide evidence that our compounds were targeting a type of cancer i.e. pancreatic. PL-45 is a primary pancreatic ductial adenocarcinoma and BxPC3 is the cell line of choice for xenograft mouse model studies.
32. Chatterjee J, Mierke DF, Kessler H. J. Am. Chem. Soc. 2006;128:15164. [PubMed]
33. Heller M, Sukopp M, Tsomaia N, John M, Mierke DF, Reif B, Kessler H. J. Am. Chem. Soc. 2006;128:13806. [PubMed]
34. Zhang X, Nikiforovich GV, Marshall GR. J. Med. Chem. 2007;50:2921. [PubMed]
35. Tyndall JD, Pfieiffer B, Abbenante G, Fairlie DP. Chem. Rev. 2005;105:793. [PubMed]
36. Viles JH, Mitchell JB, L GS, Doyle PM, Harris CJ, Sadler PJ, Thornton JM. Eur. J. Biochem. 1996;242:352. [PubMed]
37. See supplementary data for ClogP values.