Design, Synthesis, and Pharmacological Evaluation of Analogs Derived from the PLEV Tetrapeptide as Protein-Protein Interaction Modulators of Voltage-Gated Sodium Channel 1.6

Abstract

The voltage-gated Na⁺ (Na_v) channel is the molecular determinant of excitability. Disruption of protein-protein interactions (PPIs) between Na_v1.6 and fibroblast growth factor 14 (FGF14) leads to impaired excitability of neurons in clinically relevant brain areas associated with channelopathies. Here, we designed, synthesized and pharmacologically characterized new peptidomimetics based on a PLEV tetrapeptide scaffold derived from the FGF14:Na_v1.6 PPI interface. Addition of an N-terminal 1-adamantanecarbonyl pharmacophore significantly improved peptidomimetic inhibitory potency. Surface plasmon resonance studies revealed that while this moiety was sufficient to confer binding to FGF14, altering the C-terminal moiety from methoxy (21a) to π bond-containing (23a and 23b) or cycloalkane substituents (23e) abrogated the binding to Na_v1.6.Whole-cell patch-clamp electrophysiology subsequently revealed that 21a had functionally relevant interactions with both the C-terminal tail of Na_v1.6 and FGF14. Collectively, these findings support that 21a (PW0564) may serve as a promising lead to develop target selective neurotherapeutics by modulating protein-channel interactions.

Graphical Abstract

INTRODUCTION

Voltage-gated sodium (Na_v) channels, with the support of a diverse ensemble of auxiliary proteins tightly regulating their functions, serve as the primary molecular determinants of neuronal excitability.^1–3 Nine different isoforms of Na_v channels (Na_v1.1–1.9) have been reported.⁴ In addition to molecular differences in terms of their pore-forming α subunits, the isoforms diverge with respect to their localization within specific tissue types. Specifically, Na_v1.1–1.3 and 1.6 channel isoforms are localized in the central nervous system (CNS); Na_v1.7–1.9 channel isoforms are localized in the peripheral nervous system (PNS); the Na_v1.4 channel isoform is localized in skeletal muscle; and the Na_v1.5 channel isoform is localized in cardiac muscle.^{5, 6} Given this ubiquity throughout the body, it is unsurprising that Na_v channel dysfunction has been associated with a multitude of channelopathies including epilepsy,^7–9 pain,^10–12 and cardiac arrhythmias.^{13, 14} Furthermore, Na_v channel blockers are currently being used in conjunction with conventional neuropsychopharmacological agents for the treatment of bipolar disorder,¹⁵ depression,¹⁶ Alzheimer’s disease¹⁷ and schizophrenia,¹⁸ elucidating the role of Na_v channels in both neurologic and neuropsychiatric disorders.² Moreover, a number of recent reports indicate that Na_v channel dysfunction is implicated in the proliferation and migration of neoplastic cells,^19–23 the development of diabetes,²⁴ and the progression of a multitude of other humans diseases.² Given this primacy in the pathophysiology of CNS disorders, PNS disorders, cancer, and other human diseases, Na_v channels have been the targets of numerous drug discovery programs, albeit with modest success due to significant off-target effects and poor isoform selectivity.

Na_v1.6, encoded by the SCN8A gene, is one of the most abundantly distributed Na_v channel isoforms in the human CNS.^{25, 26} Foremost among the pathologies that Na_v1.6 dysfunction results in, accumulating studies have revealed that Na_v1.6 aberrations play crucial roles in the pathogenesis of epilepsy.^27–31 Studies investigating the electrical induction of status epilepticus (SE) discovered that the expression levels of Na_v1.6 were increased in medial entorhinal cortex (mEC) layer II neurons,³⁰ causing them to become hyperexcitable and conferring seizure activity, which was shown to be reversible by a Na_v1.6 inhibitor.³⁰ Correspondingly, direct evidence has demonstrated that the loss of Na_v1.6 function suppresses neuronal excitability in a SCN8A null mouse model.²⁸ Loss-of-function mutations in SCN1A, the gene that encodes Na_v1.1channels, are implicated in the pathogenesis of Dravet Syndrome (DS), a severe and currently intractable form of epilepsy. Such loss-of-function mutations in Na_v1.1channels severely impair action potential firing in hippocampal GABAergic inhibitory neurons while not perturbing the excitatory pyramidal neurons, the resultant imbalance of which gives rise to hyperexcitability. Pertinent to the present investigation, it was subsequently shown that compensatory inhibition of Na_v1.6 channels was able to restore equilibrium between excitatory and inhibitory neurons and partially rescue the epileptic phenotype in a mouse model.³² Suffice it to say, Na_v1.6 plays an important role in electrical and chemical signaling pathways in the CNS. As such, selective inhibition of Na_v1.6 could provide a novel approach for developing potential therapeutics for human CNS disorders while avoiding undesirable off-target side effects. However, designing selective small molecule inhibitors of Na_v1.6 has proven to be vexingly difficult, as there is substantial sequence homology among Na_v channel isoforms.³³ Illuminating this challenge, a series of aryl sulfonamide Na_v1.6 inhibitors was recently developed, and they showed potent anticonvulsant activity in mouse models of epilepsy.³⁴ Unfortunately, those inhibitors also displayed inhibitory activity against other Na_v channel isoforms. On account of their lack of isoform selectivity, in addition to unfavorable pharmacokinetic and toxicity profiles, these inhibitors were not selected for further investigation.³⁴

One potential approach for developing isoform selective inhibitors of Na_v1.6 is through small molecule-based rational design targeting the protein-protein interaction (PPI) interface of Na_v1.6 and its auxiliary protein fibroblast growth factor 14 (FGF14).^{35, 36} It was previously shown that binding of FGF14 to different Na_v channel isoforms markedly affected Na_v-mediated currents in a Na_v isoform-dependent fashion^37–39 and that these changes were distinct from those associated with other FGF isoforms and splice variants.^{40, 41} In addition, translational studies have increasingly illuminated an associative linkage between FGF14 dysfunction and a multitude of neuropsychiatric conditions such as schizophrenia^{16, 42} and depression.⁴³ Collectively considered, these findings suggest that the FGF14:Na_v1.6 PPI interface may serve as a promising target for pharmacological probe and drug development.

We previously identified three peptides that could inhibit FGF14:Na_v1.6 complex assembly.⁴⁴ Two of them, FLPK (1, Figure 1) and PLEV (2, Figure 1), originated from a pair of four amino acid sequences located on the β12 sheet of FGF14 at the FGF14:Na_v1.6 PPI interface, whereas the third, EYYV (3, Figure 1), originated from an amino acid sequence located on the exposed β8-β9 loop of FGF14.⁴⁴ In an effort to improve the inhibitory potency of the parental compound on FGF14:Na_v1.6 complex assembly, a series of compound 1 analogs were designed and synthesized. Of these analogs, ZL0181 (4, Figure 1)³⁶ demonstrated the best potency and selectivity, with an IC₅₀ value of 63 μM and a three-fold reduction in binding affinity toward FGF14 on account of a single amino acid mutation. Mechanism of action studies indicated that compound 4 reduced neuronal excitability by acting as a state-dependent antagonist of FGF14:Na_v1.6 complex assembly in medium spiny neurons (MSNs). Further optimization of compound 4 led to compound ZL0177 (5, Figure 1), a potent FGF14:Na_v1.6 PPI interface inhibitor, with an IC₅₀ value of 11 μM.⁴⁵ Compound 5 significantly suppressed the peak transient current density governed by Na_v1.6 channels and additionally caused a depolarizing shift in the voltage at which half the population of Na_v channels transition from a closed to conducive state.⁴⁵

Figure 1.

Chemical structures of peptides derived from the FGF14:Na_v1.6 PPI interface, and two peptide analogs based on 1. The peptidomimetics 4 and 5 have been previously described and validated as functional modulators of Na_v1.6.

In this work, new analogs derived from the parental tetrapeptide 2, a four amino acid sequence previously shown to map onto the FGF14:Na_v1.6 PPI interface and predicted to disproportionately contribute to the binding energy of the two proteins, were designed, synthesized and evaluated using an in-cell assay to characterize their effects on FGF14:Na_v1.6 complex assembly. In vitro screening of these analogs of compound 2 elucidated that addition of a 1-adamantanecarbonyl substituent to the proline residue of the tetrapeptide produced several analogs with low micromolar inhibitory potency. To orthogonally validate the primacy of this substituent, surface plasmon resonance (SPR) was employed to characterize the binding profiles of seven analogs, which revealed that only analogs featuring the N-terminal 1-admantaecarbonyl avidly bound to FGF14. Despite this substituent being necessary and sufficient to confer binding with FGF14, however, it was shown to be necessary, but not sufficient, to confer binding with Na_v1.6, as altering the C-terminal moiety from methoxy to substituents containing pi bonds and cycloalkanes produced analogs unable to bind to the C-terminal domain (CTD) of Na_v1.6. In summary, by taking advantage of this novel in-cell screening approach, along with orthogonal validation measures (e.g. a protein thermal shift (PTS) assay, SPR, and whole-cell path-clamp electrophysiology), we are able to chemically optimize analogs of the PLEV tetrapeptide in an effort to discover novel pharmacological probes and potential drug leads.

RESULTS AND DISCUSSION

Chemistry.

First, we replaced the acetyl moiety of tetrapeptide 2 with hydrogen and other substituents (R¹) leading to compounds 11, 12 and 13a-g, and their synthetic routes are outlined in Scheme 1. With protected glutamic acid (6) and (S)-2-amino-3-methylbutanamide (7) as the starting material, the intermediate 8 was obtained via a coupling reaction. Deprotection of the Fmoc group of the intermediate 8, and further coupling with compound 10 led to compound 11. Compound 12 was produced by the Cbz protection group removal of compound 11 under the condition of hydrogenation. Compounds 13a-g were obtained by reaction of compound 12 with corresponding commercially available reagents.

Scheme 1. Synthesis of Peptidomimetics 11, 12 and 13a-g^a.

^aReagents and conditions: (a) HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 77% for 8, 74% for 11. (b) HNEt₂, MeCN, rt, 30 min, quant. (c) Pd/C, H₂, MeOH, rt, overnight, 91%. (d) for 13a, benzoyl chloride, NEt₃, CH₂Cl₂, rt, overnight, 85%; for 13b, 1-isocyantoadamantane, NEt₃, CH₂Cl₂, rt, overnight, 84%; for 13c-13f, corresponding acid, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, 72~83%; for 13g, 4-fluorobenzenesulfonyl chloride, NEt₃, CH₂Cl₂, rt, overnight, 70%.

As outlined in Scheme 2, compounds 17 and 19a-k were designed to replace the acetyl group of 2 with Cbz moiety, and then keep the Cbz group intact to modify the amide moiety. The intermediate 15 was produced via a coupling reaction of compound 6 and L-valine methyl ester (14). Compound 17 was synthesized following a similar synthetic process to prepare compound 11 from compound 15. Compounds 19a-j were prepared by hydroxylation of compound 17 to produce the intermediate 18, followed by further reactions with various amines. Compound 19k was obtained by reaction of intermediate 18 with 3-bromoprop-1-ene in the presence of NaHCO_3.

Scheme 2. Synthesis of Peptidomimetics 17 and 19a-k^a.

^aReagents and conditions: (a) HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 89% for 15, 74% for 17. (b) HNEt₂, MeCN, rt, 30 min, quant. (c) (i) LiOH, MeOH, rt, overnight; (ii) 1 N NaHSO₄, 67%. (d) for 19a-19j, corresponding amine, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, 30~86%; for 19k, 3-bromoprop-1-ene, NaHCO₃, DMF, TBAI, rt, overnight, 76%.

Compounds 21a-c were designed to probe the role of R³ substituent and the synthetic procedures are outlined in Scheme 3. Synthesis starting from compound 17 led to the intermediate 20 by removal of the Cbz protection group under the condition of hydroxylation. The intermediate 20 was further converted into compounds 21a-c under the coupling condition. As depicted in Scheme 3, compounds 23a and 23b were designed by replacement of the methyl group of 21a with several alkyl moieties to determine the steric role of R⁴. Hydroxylation of compound 21c to get the acid 22, and then coupling the acid 22 with alkyl bromide or propargyl bromide to achieve the final compounds 23a and 23b.

Scheme 3. Synthesis of Peptidomimetics 21a-c, 23a and 23b^a.

^aReagents and conditions: (a) Pd/C, H₂, rt, overnight, 92%. (b) for 21a, 1-adamantanecarboxylic, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 67%; for 21b, Fmoc chloride, DIPEA, DMAP, DCM, rt, overnight, 73%; for 21c, acetyl chloride, DIPEA, DMAP, DCM, rt, overnight, 78%; (c) (i) LiOH, MeOH, rt, overnight; (ii) 1 N NaHSO₄, 80%. (d) R⁴Br, NaHCO₃, TBAI, DMF, rt, overnight, 71% for 23a; 69% for 23b.

As outlined in Scheme 4, compounds 23c-e were designed to explore additional alkyl group replacement to further determine the steric role of R⁴. The intermediate 25 was produced via a coupling reaction of compound 24 with commercially available methyl L-leucinate in a yield of 86%. Compound 26 was obtained by deprotection of the Fmoc group, followed by introducing the adamantanecarbonyl moiety. Hydrolyzation of compound 26 in the presence of LiOH led to compound 27. Coupling intermediate 6 with corresponding L-valinate⁴⁶ provided new compounds 28a-c. Compounds 23c-e were synthesized by deprotection of the Fmoc group of compounds 28a-c followed by coupling with the acid 27. As shown in Scheme 5, the tripeptide analogues 30-32 were designed in comparison with those tetra-peptidomimetics, and synthesized in a similar fashion to that of generating compound 15.

Scheme 4. Synthesis of Peptidomimetics 23c-e^a.

^aReagents and conditions: (a) methyl L-leucinate, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 86%. (b) (i) HNEt₂, MeCN, rt, 30 min, quant.; (ii) 1-adamantanecarboxylic, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 87%. (c) (ii) LiOH, MeOH, rt, overnight; (ii) 2 N HCl, 84%. (d) corresponding L-valinate, HBTU, HOBt, DIPEA, CH₂Cl₂, rt, 48 ~ 95%. (e) (i) HNEt₂, MeCN, rt, 30 min, quant.; (ii) HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 72 ~ 80%.

Scheme 5. Synthesis of Peptidomimetics 30–32^a.

^aReagents and conditions: (a) HBTU, HOBt, DIPEA, CH₂Cl₂, rt, overnight, 90% yield for 30, 90% for 31, and 84% for 32.

In-cell Pharmacological Evaluation of New Analogues of Compound 2 Using the Split-Luciferase Complementation Assay (LCA).

We have previously developed and validated an in-cell LCA that can be used to identify small molecules that modulate FGF14:Na_v1.6 complex assembly using a double stable HEK293 cell line expressing CLuc-FGF14 and CD4-Na_v1.6-NLuc recombinant proteins that, upon binding, produce luminescence in the presence of the substrate luciferin.^{47, 48} With this assay, all newly designed and synthesized peptide analogues of 2 were first screened against the FGF14:Na_v1.6 complex. Analogues were reconstituted in DMSO and administered to cells in 96-well plates at a screening concentration of 50 μM. Based on screening results, we subsequently assessed dose-dependency for the most effective compounds to further characterize in-cell activity, as well as to select candidates for follow-up studies. Compound luminescence was normalized to per plate controls (0.5% DMSO alone), and the screening results are summarized in Figure 2 and Tables 1–3.

Figure 2.

Single concentration (50 μM; n = 4 per compound) screening of the parental PLEV tetrapeptide and its analogs against the PPI between FGF14 and the CTD of Na_v1.6 using the LCA. Transfected cells were incubated with compounds for 1 hour prior to luminescence reading, and maximal luminescence for each well was normalized to per plate 0.5% DMSO controls (n = 32 per plate). Individual replicate values with mean ± SEM over at least three independent experiments are shown. A one-way ANOVA with post hoc Dunnett’s multiple comparisons test was used to determine statistical significance. F (29, 324) = 137.3; *, p<0.0005.

Table 1.

Comparison of Tetra-peptidomimetics 11, 12, 13a-g and Tripeptide Analogs 30–32

Compound	R1 (for peptidomimetics 11, 12, 13a-g)	cLogPa	RLVs (%)b
2		−0.50	79.4 ± 1.3%
11	Cbz	2.55	112.3 ± 6.1
12	-H	0.50	114.1 ± 3.2
13a		2.05	105.1 ± 6.8
13b		2.89	83.5 ± 2.8
13c		2.96	68.7 ± 2.5
13d		3.88	114.5 ± 3.0
13e		2.32	103.9 ± 5.8
13f		2.19	122.6 ± 3.0
13g		1.74	86.4 ± 2.7
30		3.10	>100%
31		1.94	>100%
32		2.63	>100%

^aclogP: http://biosig.unimelb.edu.au/pkcsm/prediction.

^bResults from screening compounds (50 μM; n = 4 replicates per compound) against the FGF14:Na_v1.6 complex using the LCA in HEK293 cells are shown as relative luminescence values (RLVs), which are calculated as percent luminescence relative to the mean of per plate controls (0.5% DMSO; n = 32 replicates per plate). Values are mean ± SEM over at least three independent experiments.

Table 3.

Comparison of Peptidomimetics 21a-c, 23a-e with Modification of R³ and R⁴

Compound	R3	R4	cLogPa	RLVs (%)b
2			−0.50	79.4 ± 1.3%
21a	1-adamantanecarbonyl	-Me	3.65	4.7 ± 0.2
21b	Fmoc	-Me	4.85	76.9 ± 4.9
21c	acetyl	-Me	1.45	95.0 ± 3.3
23a	1-adamantanecarbonyl		4.20	5.0 ± 0.7
23b	1-adamantanecarbonyl		3.65	5.1 ± 0.5
23c	1-adamantanecarbonyl		3.41	13.1 ± 0.8
23d	1-adamantanecarbonyl		4.81	33.6 ± 1.9
23e	1-adamantanecarbonyl		5.35	43.4 ± 2.4

^aclogP: http://biosig.unimelb.edu.au/pkcsm/prediction.

^bResults from screening compounds (50 μM; n = 4 replicates per compound) against the FGF14:Na_v1.6 complex using the LCA in HEK293 cells are shown as relative luminescence values (RLVs), which are calculated as percent luminescence relative to the mean of per plate controls (0.5% DMSO; n = 32 replicates per plate). Values are mean ± SEM over at least three independent experiments.

We initially investigated how various chemical substituents present at the R¹ position of the tetrapeptide altered the activity of the peptidomimetics against FGF14:Na_v1.6 complex assembly, as shown in Figure 2 and Table 1. Interestingly, these preliminary screening results revealed that a fused alkyl ring present in the R¹ position (13b and 13c) reliably maintained a similar inhibitory activity against FGF14:Na_v1.6 complex assembly as was observed for the parental PLEV (2) peptide (79.4 ± 1.3% luminescence for 2 versus 83.5 ± 2.8% and 68.7 ± 2.5% for 13b and 13c, respectively). Compound 13c was shown to be the most potent among this series with an IC₅₀ value of 49 μM (Figure 3, and Table 4). Substituents Cbz (11), hydrogen (12), long alkyl (13d), cycloalkyl (13e), and benzenesulfonyl (13g) only showed slight inhibitory effects on FGF14:Na_v1.6 complex assembly, whereas aryl substituents (13a and 13f) did not show any inhibitory activity. Moreover, tripeptide analogues truncated to PLE (compounds 30-32, Table 1) were also synthesized to further study the structure–activity relationship (SAR) of PLEV; none exhibited inhibitory activity, suggesting that the tetrapeptide scaffold is essential for activity.

Figure 3.

Determination of the potency (IC₅₀) of twelve compound 2 analogs. Utilizing the LCA, peptidomimetics were tested at eight concentrations (range: 0.25 – 100 μM; n = 4 replicates per concentration). Data are shown as mean percent luminescence (relative to on-plate 0.5% DMSO controls) values ± SEM. GraphPad Prism 8 software was used for non-linear curve fitting to estimate the half maximal inhibitory concentration for compound 2 analogs, and their corresponding IC₅₀ and efficacy values are shown in Table 4.

Table 4.

IC₅₀ Values and Efficacy of Top Peptidomimetics.^a

Compound	IC50 (μM)	Efficacy (%)
13c	49.0 ± 11.3	82.1
17	22.4 ± 1.1	93.3
19k	15.4 ± 0.3	90.7
19e	33.4 ± 2.4	96.9
21a	20.4 ± 0.6	96.1
23a	11.5 ± 0.6	95.5
23b	13.8 ± 1.4	96.6
23c	25.1 ± 9.6	91.4
23d	33.9 ± 6.4	92.1
23e	35.3 ± 4.4	69.9
23f	13.3 ± 0.6	82.3
23g	22.5 ± 6.1	83.3

^aPotency (estimated IC₅₀, value ± SEM; μM) and efficacy (percent inhibition, calculated as the inverse of normalized luminescence at the bottom plateau) values for twelve compound 2 analogs were determined using the dose response curves shown in Figure 3.

Subsequently, chemical optimization to increase the lipophilicity of 2 by replacing the amino group with an alkyl substituted amine or ester at the R² position provided compounds 17 and 19a-k. As shown in Figure 2 and Table 2, the LCA results revealed that compounds 17 and 19k with an ester occupying the R² position demonstrated significantly improved efficacy in terms of inhibiting the PPI between FGF14 and the CTD of Na_v1.6.. Compounds with secondary amine substituents occupying the R² position (19a, 19b, 19e, and 19i) exhibited diminished efficacy in terms of inhibiting the PPI between FGF14 and the CTD of Na_v1.6 relative to 17 and 19k, with the singular exception of 19e. Notably, of five compounds with primary amine substituents occupying their R² position, four molecules (19c, 19d, 19g and 19j) potentiated FGF14:Na_v1.6 complex assembly, whereas one, 19h, displayed minimal inhibitory activity. Overall, for compounds with modifications at R², we assessed dose-dependency for 17, 19e, and 19k based on their pronounced screening effects. As shown in Figure 3 and Table 4, compounds with an ester at the R² position (17 and 19k) exhibited greater potency than those with a secondary amine (19e) (IC₅₀ values of 22.4 μM and 15.4 μM, respectively, vs. 33.4 μM for 19e). Conversely, the latter compound (19e) exhibited the greatest maximal effect (efficacy values of 93.3%, 90.7%, and 96.9% luminescence for 17, 19k, and 19e, respectively).

Table 2.

Comparison of Peptidomimetics 17 and 19a-k with Modifications at R²

Compound	R2	cLogPa	RLVs (%)b
2		−0.50	79.4 ± 1.3%
17	-OMe	3.23	39.8 ± 3.3
19a		2.92	72.0 ± 4.4
19b		3.93	92.3 ± 3.6
19c		2.81	103.2 ± 3.6
19d		4.38	133.8 ± 2.5
19e		3.69	14.0 ± 1.2
19f		2.27	111.9 ± 1.0
19g		4.51	117.2 ± 3.0
19h		5.15	81.5 ± 3.2
19i		2.84	54.2 ± 1.8
19j		2.17	118.1 ± 2.3
19k	-Oallyl	3.80	11.9 ± 0.7

^aclogP: http://biosig.unimelb.edu.au/pkcsm/prediction.

^bResults from screening compounds (50 μM; n = 4 replicates per compound) against the FGF14:Na_v1.6 complex using the LCA in HEK293 cells are shown as relative luminescence values (RLVs), which are calculated as percent luminescence relative to the mean of per plate controls (0.5% DMSO; n = 32 replicates per plate). Values are mean ± SEM over at least three independent experiments.

Based upon the preliminary screening results of the first two batches of compound 2 analogs, we discerned that fused alkyl ring and alkoxyl substituents in the R¹ and R² locales, respectively, conferred compounds with enhanced inhibitory properties against FGF14:Na_v1.6 complex assembly. Given this, we designed and synthesized a third batch of compound 2 analogs with a constant fused alkyl ring at the R³ position as an optimal moiety and a variable alkoxyl substituent at the R⁴ position, which produced 21a, 23a, 23b, 23c, 23d, and 23e (Figure 2 and Table 3). The in-cell screening of this new series of compound 2 analogs revealed that a fused alkyl ring and a methoxy in the R³ and R⁴ locales, respectively (21a), resulted in marked disruption of the PPI between FGF14 and the CTD of Na_v1.6 (IC₅₀ = 20.4 μM, Efficacy = 96.1%, Figure 3 and Table 4). Replacing the 1-adamantanecarbonyl of compound 21a with Fmoc and acetyl led to compound 21b and 21c, respectively, which showed markedly reduced inhibitory activity, recapitulating our finding that a fused alkyl ring substituent at the R³ position is essential in conferring these peptidomimetics with high potency and efficacy (Figure 2 and Table 3). Replacement of the methyl group of compound 21a with allyl and propargyl led to compounds 23a and 23b, respectively, both of which retained similar RLVs compared to 21a (Figure 2 and Table 3). As depicted in Figure 3 and Table 4, subsequent dose-dependency studies of compounds 23a and 23b revealed ~2-fold increases in potency when compared to compound 21a (IC₅₀ = 20.4 μM), with IC₅₀ values of 11.5 μM and 13.8 μM, respectively. Ethyl (23f) and propyl (23g) ester analogs of 21a yielded a moderate loss of efficacy (82.3 and 83.3%, respectively), although 23f did exhibit moderately improved potency (IC₅₀ = 13.3 μM) relative to 21a.

Counter-Screening of Top FGF14:Na_v1.6 Inhibitors Using Full-length Luciferase and Toxicity Assays.

To ensure that the putative effects of compound 2 analogs on the PPI between FGF14 and the CTD of Na_v1.6 did not stem from direct activity against the luciferase enzyme itself or cell toxicity, twelve analogs shown to inhibit FGF14:Na_v1.6 complex assembly during the initial screening were tested against the native full-length luciferase (FLL) enzyme and using the CellTiter-Blue® Cell Viability (CTB) assay, respectively (Figure 4). These investigations demonstrated that the most potent and efficacious compound 2 analogs neither directly inhibited the FLL (Figure 4A) nor demonstrated cell toxicity (Figure 4B).

Figure 4.

Counter-screening of top 12 FGF14:Na_v1.6 inhibitors using luciferase and toxicity assays. (A) After transfecting HEK293 cells with the full-length photinus pyralis luciferase enzyme, cells were incubated with DMSO (0.5%; n = 32 replicates per plate) or a compound 2 analog (25 μM; n = 4 replicates per plate) for one hour, after which the LCA was performed as previously described. F (12, 83) = 0.4481; p > 0.05. (B) To assess the cellular toxicity of compound 2 analogs, the CellTiter Blue reagent was added to each well following luminescence readings in (A). Detection of fluorescence was performed after 18 h of incubation with the reagent for detecting cellular toxicity. F (12, 83) = 1.817; p > 0.05. Data are mean ± SEM. A one-way ANOVA with post hoc Dunnett’s multiple comparisons test was employed to determine statistical significance.

Determination of Peptidomimetic Binding by PTS and SPR.

Based on the LCA results (Figure 3), the peptidomimetics that demonstrated promising potency (low IC₅₀), efficacy (maximal %inhibition), and dose dependency (curve shape) were moved forward to the next stage, yielding a set of seven active compounds (19k, 19e, 21a, 23a, 23b, 23d, 23e). The goal was to determine whether these peptidomimetics bind to FGF14 and/or the CTD of Na_v1.6, as predicted based on homology modeling and docking studies. Recombinant human FGF14 and Na_v1.6 CTD proteins were purified as described previously and in the Experimental Section.³⁶

First, compounds were screened for binding to human FGF14 or Na_v1.6 C-terminal tail proteins using the PTS assay in 96-well plates. Analysis of protein thermal stability is an effective in vitro technique for assessing binding between ligands and protein of interest, as determined through changes in melting temperature (ΔT_M).⁴⁹ Proteins have a specific thermal stability (melting temperature), and ligand binding can significantly shift this stability to become either more or less stable depending on the new conformational state. As the protein unfolds, the exposed hydrophobic regions interact with the reaction dye to produce fluorescence. The top seven compounds were tested against both FGF14 and Na_v1.6 C-terminal tail recombinant protein separately. Treatments were normalized to the average T_M of the per plate FGF14 or Na_v1.6 protein only alone control wells (n = 8 per plate). As determined by a change in melting temperature of ≥ ± 2 °C, four of the compounds were found to bind to FGF14, one of which (compound 21a) was also found to bind the Na_v1.6 C-terminal tail (Figures 5 and 6; Table 5). The greatest ΔT_M was observed with compound 21a (−4.64 °C for Na_v1.6; −2.27 °C for FGF14), followed by compound 23a (−4.55 °C), 19e (3.38 °C), and 23e (−2.21 °C).

Figure 5.

Determination of protein:ligand binding of compound 2 analogs to FGF14 and the CTD of Na_v1.6 using protein thermal shift. The top 7 peptidomimetics were screening for binding to purified FGF14 or Na_v1.6 C-terminal tail protein in 96-well plates (n = 4 replicates per compound) using the fluorescence-based PTS assay. Hits were defined as those compounds that resulted in a change in protein melting temperature (ΔT_M) by ≥ ± 2 °C compared to protein alone and are highlighted as either red (hits against FGF14) or blue (hits against Na_v1.6). Individual replicates and the mean ΔT_M is shown for each condition.

Figure 6.

Quantification of the binding affinity (K_D) of compound 2 analogs toward FGF14 and Na_v1.6 protein using SPR. Increasing concentrations of compound 2 analogs (0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, and 200 μM) were flown over purified FGF14 (left) or Na_v1.6 (right) protein covalently attached to CM5 chips. Association and dissociation times for each sample were held at 120 and 150 s, respectively. The kinetics of each protein:ligand interaction were analyzed by fitting the kinetic responses to the simplest Langmuir 1:1 interaction model (K_D = k_off/k_on). The binding sensorgrams (left) and steady-state saturation plots (right) are represented for each ligand against each protein. The corresponding equilibrium dissociation constants (K_D), as well as association (k_on) and dissociation (k_off) rates, are shown in Table 6.

Table 5.

Determination of Protein:ligand Binding of Compound 2 Analogs to FGF14 and Na_v1.6 by Thermal Shift.

	ΔTM (°C)*
Compound	FGF14	Nav1.6
19k	0.67 ± 0.05	−0.87 ± 0.48
19e	3.38 ± 0.08	−0.48 ± 0.79
21a	−2.27 ± 0.18	−4.64 ± 0.43
23a	−4.55 ± 0.13	0.04 ± 0.07
23b	−1.85 ± 0.40	−0.15 ± 0.12
23d	−0.89 ± 0.15	−0.13 ± 0.20
23e	−2.21 ± 0.39	0.43 ± 0.47

^*Change in melting temperature (°C) was calculated relative to per plate FGF14 and Na_v1.6 protein alone controls.

To quantitatively assess the binding affinity of peptidomimetics, SPR experiments with 19k, 19e, 21a, 23a, 23b, 23d, and 23e were performed in duplicate and each compound flown at 11 concentrations over FGF14 and Na_v1.6 C-terminal tail protein immobilized to CM5 sensor chips. The SPR experiments serve as both an orthogonal approach (in conjunction with PTS) of assessing protein:ligand binding, as well as to determine accurate binding affinities (K_D). With the exception of the 19 series, the remaining compound 2 analogs bound strongly to FGF14 with affinities ranging from 1.3 to 14.7 μM (Figure 6; Table 6). Compound 19k demonstrated negligible binding by both PTS and SPR. Conversely, compound 19e stabilized FGF14 protein by PTS, but exhibited relatively poor binding affinity by SPR (K_D = 188.9 μM). Given their in-cell potency toward disrupting FGF14:Na_v1.6 complex formation, these relatively variable in vitro phenotypes for the 19 series compounds may arise from differences in assay environments, and as such were put on hold for future studies.

Table 6.

Quantitative Determination of Protein:ligand Binding of Compound 2 Analogs to FGF14 and Na_v1.6 by SPR.

Compound	FGF14			Nav1.6 C-terminal tail
	KD* (μM)	kon (M−1 s−1)	koff (s−1)	KD* (μM)	kon (M−1 s−1)	koff (s−1)
19k	ND	ND	ND	> 200	2.57 × 105	4.49 × 10−3
19e	188.9 ± 3.7	4.61 × 102	9.91 × 10−4	ND	ND	ND
21a	4.05 ± 0.17	4.66 × 103	9.33 × 10−3	16.43 ± 2.05	6.23 × 104	3.57 × 10−3
23a	1.31 ± 0.37	1.79 × 104	9.32 × 10−3	> 200	6.16 × 106	2.73 × 10−3
23b	14.72 ± 1.40	2.58 × 104	2.69 × 10−7	> 200	1.26 × 105	1.45 × 10−3
23d	6.94 ± 1.08	1.62 × 103	5.80 × 10−3	> 200	8.29 × 105	7.94 × 10−3
23e	7.02 ± 1.20	7.35 × 103	1.39 × 10−2	> 200	7.16 × 105	1.70 × 10−4
23f	9.47 ± 1.22	2.17 × 102	2.05 × 10−2	67.34 ± 7.3	2.64 × 103	1.22 × 10−2
23g	11.39 ± 2.15	2.89 × 102	2.04 × 10−2	90.14 ± 10.1	3.40 × 102	4.92 × 10−2

^*The equilibrium dissociation constants (K_D) were calculated using data represented in Figure 6. The K_D shown for each protein:ligand interaction is an average of the values calculated using the simplest Langmuir 1:1 interaction model (K_D = k_off/k_on) and the steady-state saturation model. ND, non-determinable.

Most compounds bound poorly or not at all to Na_v1.6 C-terminal tail (Table 6), suggesting that these compounds would have a mechanism of inhibiting FGF14 binding with Na_v1.6 via binding and sequestering FGF14 at the interface involved in mediating binding to Na_v1.6. Interestingly, only compound 21a and its ethyl and propyl ester analogs (23f and 23g) had appreciable binding to Na_v1.6, with the bulkier groups of 23f and 23g resulting in lower affinities. Conversely, the other compounds demonstrated association and dissociation rates that were too fast or slow, respectively, for the instrument to reliably measure, indicating weak or negligible binding. This finding is in agreement with the PTS results showing that only compound 21a changed the thermal stability of Na_v1.6. Based upon the collective findings of the PTS and SPR investigations, compound 21a was selected for subsequent functional validation studies based upon the discovery that it bound to both FGF14 and the CTD of Na_v1.6, as well as its heightened binding affinity toward the latter relative to its ethyl and propyl ester analog counterparts.

Having characterized 21a’s protein:ligand interactions with FGF14 and the CTD of Na_v1.6 individually, we next assessed its effects on the PPI between FGF14 and the CTD of Na_v1.6 (Figure 7). Using a sensor chip with Nav1.6 bound, increasing concentrations of FGF14 either alone or in complex with 21a were flown over the sensor chip surface. FGF14 exhibited high affinity binding toward Na_v1.6 C-terminal tail (K_D = 228.5 nM), but this binding was substantially reduced in the presence of 21a (K_D = 1419 nM). These results indicate that 21a is directly capable of binding FGF14, as well as hindering binding of FGF14 to Na_v1.6. Given 21a’s appreciable binding affinities toward both FGF14 and the CTD of Na_v1.6, its ability to reduce FGF14’s binding to Na_v1.6 likely arises from two pharmacodynamically distinct processes: 1) direct binding to the CTD of Na_v1.6 and resultant occupancy of the FGF14 interaction site and 2) binding to FGF14 that causes the protein to undergo a conformational change that renders it unable to access its interaction site on the CTD of Na_v1.6. Despite being pharmacodynamically divergent, it is expected that these mechanisms will have synergetic effects and collectively contribute to disrupting the PPI between FGF14 and the CTD of Na_v1.6.

Figure 7.

Compound 21a disrupts FGF14 binding to the CTD of Na_v1.6 as determined by SPR. Increasing concentrations of FGF14 (15 – 1500 nM) were flown over Na_v1.6 protein bound to CM5 chips. Association and dissociation times for each sample were held at 120 and 150 s, respectively. Kinetic analysis of each ligand/analyte interaction was obtained by fitting the response data to the simplest Langmuir 1:1 interaction model (K_D = k_off/k_on). Representative binding sensorgrams (left) and steady-state saturation plots (right) are shown for each compound against each protein. The resulting equilibrium dissociation constants (K_D), as well as kinetic association (k_on) and dissociation (k_off) rates are provided in Table 7. Steady-state saturation plot for comparison of FGF14 alone (WT, black) versus FGF14 in complex with 21a (10 μM; blue) binding to Na_v1.6 with response units (RU) relative to the maximal binding response of FGF14 alone.

Electrophysiological Evaluation of Lead Compound.

To elucidate direct and indirect mechanisms by which 21a could confer modulatory effects on Na_v1.6 channel kinetics, as well as characterize its selectivity for Na_v channel isoforms in the CNS, the compound was subjected to extensive functional evaluation. To assess the functional consequences of a direct mechanism of Na_v1.6 modulation mediated via 21a, HEK293 cells that stably express human Na_v1.6 (HEK-Na_v1.6) channels were incubated for at least 30 min with either 0.1% DMSO (control) or 10 μM 21a. After at least 30 min, effects of 21a on the kinetics of Na_v1.6 channels were investigated using whole-cell patch-clamp electrophysiology (Figure 8 and Table 8). During these electrophysiological studies, carried out in the absence of FGF14, treatment of HEK-Na_v1.6 cells with 10 μM 21a induced phenotypes similar to those observed in HEK-Na_v1.6 cells co-expressing FGF14, including suppression of peak current density (−50.1 ± 3.1 pA/pF versus −25.8 ± 2.3 pA/pF for DMSO and 21a, respectively; Figures 8C and 8D), a depolarizing shift of V_1/2 of activation (−25.4 ± 1.0 mV versus −16.9 ± 0.67 mV for DMSO and 21a, respectively; Figures 8E and 8F), and a depolarizing shift in the V_1/2 of steady-state inactivation (−62.0 ± 1.3 mV versus −55.2 ± 0.85 mV for DMSO and 21a, respectively; Figures 8G and 8H).^{36, 50} Whereas 21a conferred these modulatory effects on HEK-Na_v1.6 cells, no changes in the kinetic properties of Na_v1.1 channels (HEK-Na_v1,1 cells; Supporting Information, Figure S2 and Table S1) or Na_v1.2 channels (HEK-Na_v1.2 cells; Supporting Information, Figure 3S and Table S2) were induced by the compound. Collectively, these investigations demonstrate a direct and selective mechanism by which 21a modulates the kinetic properties of Na_v1.6 channels.

Figure 8.

Electrophysiological evaluation of 21a in HEK-Na_v1.6 cells in the absence of FGF14. (A) Representative traces of transient Na⁺ currents recorded from HEK-Na_v1.6 treated with 0.1% DMSO (black) or 10 μM 21a (blue) in response to varying voltage stimuli ranging from −100 mV to + 60 mV(inset). (B) Comparison of tau of fast inactivation at −10 mV between the indicated experimental groups. (C) Current-voltage relationship for HEK-Na_v1.6 treated with 0.1% (black) or 10 μM 21a. (D) Comparison of peak current density among the indicated experimental groups. (E) Conductance-voltage relationship for HEK-Na_v1.6 treated with 0.1% DMSO (black) or 10 μM 21a. (F) Comparison of V_1/2 of Na_v1.6 channel activation between the indicated experimental groups. (G). Comparison of the voltage-dependence of steady-state inactivation between HEK-Na_v1.6 treated with 0.1% DMSO (black) or 10 μM 21a. (H) Comparison of V_1/2 of the steady-state inactivation of Na_v1.6 channels between the indicated experimental groups. (I) Effects of 0.1% DMSO (black) and 10 μM 21a (blue) on the long-term inactivation (LTI) of Na_v1.6 channels. LTI was characterized by plotting the fraction of Na_v1.6 channels available as a function of depolarization cycle. See experimental section for additional information describing electrophysiological protocols employed and corresponding data analyses. (J) Bar graph summary of (I). Results are expressed as the mean ± SEM. A Student’s t-test was used to determine statistical significance. . **, p < 0.005; ***, p < 0.0005.

Table 8.

Functional modulation of Na_v1.6 in the absence of FGF14 by 10 μM 21a compared to 0.1% DMSO.^a

Condition	Peak Current Density (pA/pF)	Tau of Fast Inactivation (ms)	V1/2 of Activation (mV)	V1/2 of Steady-State Inactivation (mV)	Long Term Inactivation (Depolarization Cycles 2–4)
					2	3	4
DMSO	−50.1 ± 3.1 (9)	1.22 ± 0.07 (9)	−25.4 ± 1.0 (7)	−62.0 ± 1.3 (5)	0.84 ± 0.03	0.76 ± 0.05	0.75±0.05 (6)
21a	−25.8 ± 2.3*** (10)	1.45 ± 0.15 (10)	−16.9 ± 0.67***(10)	−55.2 ± 0.85 (8)**	0.87 ± 0.02	0.76 ± 0.03	0.70 ± 0.03 (6)

^aSummary of the electrophysiological evaluation of 21a against Na_v1.6 in the absence of FGF14. Results are expressed as the mean ± SEM. The number of independent experiments is shown in parentheses. A Student’s t-test was used to determine statistical significance.

^**,p < 0.005;

^***,p < 0.0005.

To investigate the extent to which 21a’s modulatory effects on Na_v1.6-mediated currents are graded, HEK-Na_v1.6 cells were also incubated with 30 μM 21a prior to electrophysiological experimentation (Supporting Information, Figure 4S A–C, blue and Table S3). As this concentration is nearly double the compound’s K_D toward the C-terminal tail of Na_v1.6, Na_v1.6 channels are expected to be approaching saturation under these experimental conditions. These investigations revealed that although there was a decrease in peak current density in HEK-Na_v1.6 cells treated with 30 μM (−17.9 ± 0.55 pA/pF; n = 8) compared to 10 μM (−25.8 ± 2.3 pA/pF; n = 10) 21a, the change was not statistically significant (p = 0.0687), suggesting that at 10 μM, 21a’s efficacy in terms of suppressing Na_v1.6-mediated currents is beginning to reach a plateau. Lastly, to validate that compound binding to the CTD of Na_v1.6 determined by SPR is predictive of functional modulation of Na_v1.6 mediated currents in the HEK-Na_v1.6 cell system, electrophysiological studies were also performed on HEK-Na_v1.6 cells incubated with 30 μM 23a. Corroborating the SPR results, which showed that analogs with π bond containing C-terminal substituents showed negligible binding to the C-terminal tail of Na_v1.6, Na_v1.6-mediated currents were unaffected in HEK-Na_v1.6 cells treated with 30 μM 23a, an analog with a C-terminal allyl substituent (Supporting Information, Figure 4S A–C, red and Table S3). As occupancy of the FGF14 interaction site of the C-terminal tail of Na_v1.6 is likely necessary for appreciable modulation of the FGF14:Na_v1.6 PPI, only 21a was pursued in subsequent investigations.

Having demonstrated a direct mechanism by which 21a could modulate kinetic properties of Na_v1.6 channels, which is expected (based upon SPR studies) to be conferred via direct binding to the CTD of Na_v1.6, we next sought to characterize whether 21a could exert modulatory effects by binding to FGF14 and disrupting its PPI with Na_v1.6. To do so, HEK-Na_v1.6 cells transiently transfected with FGF14 were incubated for at least 30 min with either 0.1% DMSO (control) or 10 μM 21a, after which the modulatory effects of the compound were again evaluated using whole-cell patch-clamp electrophysiology (Figure 9 and Table 9). Whereas in the absence of FGF14 21a induced phenotypes similar to those observed in HEK-Na_v1.6 cells co-expressing FGF14, in the presence of FGF14, 21a was shown to reverse many of the previously shown modulatory effects of FGF14 on Na_v1.6 channel kinetics.^{36, 50, 51} For example, 21a partially reversed FGF14-mediated suppression of peak current density (−19.3 ± 1.3 pA/pF versus −38.8 pA/pF for DMSO and 21a, respectively; Figures 9C and 9D), FGF14-mediated increase of tau of fast inactivation (1.58 ± 0.18 ms versus 1.00 ± 0.08 ms for DMSO and 21a, respectively; Figure 9B), and the depolarizing shift in V_1/2 of steady-state inactivation induced by FGF14 (−55.1 ± 2.0 mV versus −65.6 ± 0.66 mV for DMSO and 21a, respectively; Figures 9G and 9H). Additionally, in the presence of FGF14, 21a induced long-term inactivation of Na_v1.6 (Figures 9I and 9J) at a level comparable to that observed in the Na_v1.6 channel alone condition (Figures 8I and 8J). This phenotype could be reconciled with more complex interactions of the compound with the FGF14:Na_v1.6 channel complex. Taken together with the findings of the electrophysiological studies carried out in the absence of FGF14, these studies provide evidence for functionally relevant interactions of 21a with both FGF14 and the C-terminal tail of Na_v1.6, which collectively confer it with the ability to disrupt the PPI between the two proteins. Such modulatory effects suggest 21a may suppress neuronal excitability by reducing the number action of potentials and possibly shifting the action potential voltage threshold to a more depolarized level. However, given the heterogeneity of accessory modulatory proteins of the Na_v channel complex in the native system, more complex phenotypes with mechanisms of partial antagonism or allosteric modulation could be observed in the brain.

Figure 9.

Compound 21a reverses regulatory effects of FGF14 on Na_v1.6-mediated currents. (A) Representative traces of transient Na⁺ currents recorded from HEK-Na_v1.6 co-expressing FGF14 treated with 0.1% DMSO (black) or 10 μM 21a (blue) in response to varying voltage stimuli ranging from −100 mV to + 60 mV (inset). Note the phenotypic appearance of Na⁺ currents with a slow entry of the channel into fast inactivation in the presence of FGF14 (panel A, black), a phenotype that was reversed in the presence of compound 21a. (B) Comparison of tau of fast inactivation at −10 mV between the indicated experimental groups. (C Current-voltage relationship for HEK-Na_v1.6-FGF14 cells treated with 0.1% DMSO (black) or 10 μM 21a. (D) Comparison of peak current density between the indicated experimental groups. (E) Conductance-voltage relationship for HEK-Na_v1.6-FGF14 cells treated with 0.1% DMSO (black) or 10 μM 21a. (F) Comparison of V_1/2 of Na_v1.6 channel activation between the indicated experimental groups. (G) Effects of 0.1% DMSO (black) and 10 μM 21a on the voltage-dependence of V_1/2 of Na_v1.6 channel steady-state inactivation. (H) Comparison of V_1/2 of Na_v1.6 channel steady-state inactivation between the indicated experimental groups. (I) Effects of 0.1% DMSO (black) and 10 μM 21a on LTI of Na_v1.6 channels. LTI was characterized by plotting the fraction of Na_v1.6 channels available as a function of depolarization cycle. See experimental section for additional information regarding electrophysiological protocols employed and the corresponding data analyses. (J) Bar graph summary of (I). Results are expressed as the mean ± SEM. A Student’s t-test was used to determine statistical significance. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Table 9.

Functionally relevant interaction of 21a with FGF14 reverses many of the regulatory effects FGF14 confers on Na_v1.6.^a

Condition	Peak Current Density (pA/pF)	Tau of Inactivation (ms)	V1/2 of Activation (mV)	V1/2 of Inactivation (mV)	Long Term Inactivation (Depolarization Cycles 2–4)
					2	3	4
DMSO	−19.3 ± 1.3 (12)	1.58 ± 0.18 (7)	−21.4 ± 1.1 (6)	−55.1 ± 2.0 (4)	1.00 ± 0.02	0.99 ± 0.04	0.98 ± 0.06 (8)
21a	−38.8 ± 1.9*** (12)	1.00 ± 0.08* (6)	−21.6 ± 0.74 (10)	−65.6 ± 0.66 (6)**	0.86 ± 0.02*	0.81 ± 0.01*	0.75 ± 0.02* (8)

^aSummary of the electrophysiological evaluation of 21a against HEK-Na_v1.6 cells co-expressing FGF14. Results are expressed as the mean ± SEM. The number of independent experiments is shown in parentheses. A Student’s t-test was used to determine statistical significance.

^*,p <0.05;

^**,p < 0.005;

^***,p < 0.0005.

Molecular Docking Study Based on FGF14:Na_v1.6 Homology Model.

To further elucidate the SAR and binding modes of these peptidomimetics, we docked the newly identified peptidomimetics to the previously identified druggable pocket of the FGF14 homology model using the Schrödinger Drug Discovery Suite program.^{42,36, 44} Based on the strong experimental results discussed above, we show the predicted binding pose of analog 21a as an example case. The docking results indicated that compound 21a has energetically favorable interactions and readily docks into the pocket of the residues of β8, β9 and β12. As shown in Figure 10A and S5, the N-terminus (adamantine group) of 21a is predicted to form hydrophobic interactions with residues Val160, Leu202, Pro203 and Pro205, while the C-terminus of 21a forms hydrophobic interactions with residues loop of Met210, Gln72, Leu111 and Ile112. The hydrophobic interactions between the N-terminal adamantine group with Val160 may be of particular importance, as previous studies have identified this site as a hot-spot for interactions between FGF14 and Na_v1.6.⁵⁰ Taken together with SPR, which demonstrated significant binding for all compounds except those without an N-terminal adamantine group (19e, 19k), these findings suggest that the highly hydrophobic adamantine moiety may confer potent activity and FGF14 binding via interactions with V160.

Figure 10.

Binding modes and molecular docking of compound 21a with FGF14:Na_v1.6. The FGF14:Na_v1.6 complex homology model was generated by taking advantage of FGF13:Na_v1.5 (PDB code: 4DCK) crystal structure as a template. (A) Docking of compound 21a (magenta) into the pocket of FGF14. Important residues of the targeted protein are drawn as sticks. Hydrogen bonds are shown as dashed black lines. (B) Overlay of Na_v1.6 with docked compound 21a and FGF14. The C-terminal tail of the Na_v1.6 and FGF14 are shown in orange and green, respectively. The key amino acids of FGF14 Lys74, Arg117, Glu152, Val160, Leu202, Pro203 and Pro205 are shown as green sticks. Critical amino acids of the C-terminal tail of Na_v1.6 E1884, D1846, T1887, I1886 and R1892 are shown as red sticks.

Additionally, compound 21a forms three hydrogen bonds with residues Lys74, Arg117 and Glu152, a binding mode that may explain the significant loss of activity following truncation of the tetrapeptide into tripeptides 30-32 (Table 1). Moreover, these docking results are also in full agreement with our SAR studies demonstrating that large hydrophobic moieties at the N-terminus are essential in conferring improved potency. Overlaying of 21a with the FGF14:Na_v1.6 PPI interface homology model indicates that 21a mimics the crucial regions at the interface of FGF14 (Figure 10B) to impair the formation of the FGF14:Na_v1.6 complex.^{45, 50} Taken together, the docking studies revealed a multitude of predicted binding interactions between 21a and both FGF14 and the FGF14:Na_v1.6 PPI interface. This docking analysis offers a refined understanding of the ligand-target interactions and provides pivotal insights into measures that could be employed to chemically optimize these peptidomimetics to further improve their binding affinity toward the FGF14:Na_v1.6 PPI interface.

CONCLUSIONS

In summary, a series of novel peptidomimetics derived from the previously identified lead parental tetrapeptide, compound 2, has been designed, synthesized, and pharmacologically evaluated as modulators of FGF14:Na_v1.6 complex assembly. Among this series, several peptidomimetics including 17 (PW0164), 21a (PW0564), and 23a (PW0927) displayed low micromolar inhibitory potency and appreciable efficacy, robust effects that were demonstrated to not merely be artifacts of directly inhibiting full-length luciferase or causing cellular toxicity. Biophysical evaluation of compound 21a demonstrated that it strongly bound to both FGF14 and Na_v1.6 C-terminal tail (K_D values of 4.1 μM and 16.4 μM, respectively), and is capable of appreciably disrupting binding between these two proteins. Furthermore, evaluation of 21a using whole-cell patch-clamp electrophysiology revealed potent functional modulation of Na_v1.6 channel kinetics, which varied depending on the presence of FGF14. FGF14-mediated suppression of Na_v1.6 mediated currents is a well-studied phenomenon,^{36, 50} and in the presence of FGF14, 21a increased Na_v1.6 currents despite an inverse activity (current suppression) versus the channel alone. This striking phenotype suggests that 21a is capable of acting as a buffer toward the Na_v1.6 channel to prevent extreme changes in activity; while suppressing basal Na_v1.6 activity, it also prevents excess suppression that could be induced by pathologic changes in levels of FGF14. Crucially, among the principal Na_v CNS isoforms, 21a was confirmed to be selective toward only the Na_v1.6 channel, suggesting that this compound is an ideal lead for further neurotherapeutic and probe development studies in the future, as specificity and selectivity are vital for limiting side-effects. Docking studies revealed important hydrogen bonding and hydrophobic interactions between 21a and FGF14 at residues previously shown to mediate interactions with Na_v1.6,⁵⁰ and an overlay analysis demonstrated that 21a mimics a loop crucial to FGF14’s interaction with the intracellular C-terminal tail of Na_v1.6, a mechanism that may confer the ability to inhibit the PPI. Collectively, these findings suggest that 21a may serve as a promising scaffold from which to develop highly specific neurotherapeutics targeting the PPIs in the CNS.

EXPERIMENTAL SECTION

General Chemistry Information.

All commercially available starting materials and solvents were reagent grade, and used without further purification unless otherwise specified. Reactions were performed under a nitrogen atmosphere in dry glassware with magnetic stirring. Preparative column chromatography was performed using silica gel 60, particle size 0.063–0.200 mm (70–230 mesh, flash). Analytical TLC was carried out employing silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the developed chromatograms was performed with detection by UV (254 nm) and 0.05% wt. KMnO₄ (aq.). NMR spectra were recorded on a Bruker-300 (¹H, 300 MHz; ¹³C, 75 MHz) spectrometer. ¹H and ¹³C NMR spectra were recorded with TMS as an internal reference. Chemical shifts were expressed in ppm, and J values were given in Hz. High-resolution mass spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the following: Nano ESI spray voltage was 1.8 kV; Capillary temperature was 275 °C and the resolution was 60,000; Ionization was achieved by positive mode. All biologically evaluated compounds are > 95% pure.

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (8).

A solution of Fmoc-Glu(O^tBu)-OH (6) (1.3 g, 3 mmol) and H-Val-NH₂•HCl (7) (459 mg, 3 mmol) in 20 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Then, HOBt (459 mg, 3 mmol), HBTU (2.3 g, 6 mmol) and DIPEA (2 mL, 12 mmol) were added to the mixture solution at 0 °C. The mixture solution was stirred at room temperature overnight. When the reaction reached the end point (monitored by TLC), the solution was washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then the solution was concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to afford compound 8 (1.3 g, 77%) as a white solid. ¹H NMR (300 MHz, Methanol-d₄) δ 7.80 (d, J = 7.5 Hz, 2H), 7.65 (d, J = 6.1 Hz, 2H), 7.35 (dt, J = 24.0, 7.7 Hz, 4H), 4.45 – 4.33 (m, 2H), 4.23 (dt, J = 9.1, 4.5 Hz, 3H), 2.40 – 2.23 (m, 2H), 2.17 – 2.02 (m, 2H), 1.88 (s, 1H), 1.45 (d, J = 2.3 Hz, 9H), 0.96 (td, J = 7.0, 2.3 Hz, 6H). ¹³C NMR (75 MHz, Methanol-d₄) δ 172.74, 172.60, 157.05, 141.17, 127.37, 126.76, 124.78, 119.51, 80.44, 66.61, 58.15, 54.30, 47.52, 47.32, 47.03, 47.01, 46.75, 31.33, 30.63, 26.95, 18.34, 16.95.

Benzyl (S)-2-(((S)-1-(((S)-1-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-5-(tert-butoxy)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (11).

Compound 8 was added to the solution of HNEt₂ (1 mL) and MeCN (3 mL), and the mixture solution was stirred at room temperature for 1 h. After the reaction completed (monitored by TLC), the solvent was removed under vacuum to generate compound 9 as a colorless oil. To a solution of compound 9 in 20 mL of dry CH₂Cl₂, compound Cbz-Pro-Leu-OH (10) (724, 2 mmol) was added and the mixture solution was cooled to 0 °C with an ice bath. Then, HOBt (270 mg, 2 mmol), HBTU (1.5 g, 4 mmol) and DIPEA (1.3 mL, 8 mmol) were added to the solution at 0 °C. The mixture solution was stirred at room temperature overnight. When the reaction reached the end point (monitored by TLC), the solution was washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then the solution was concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain compound 11 (0.96 g, 74%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.16 – 7.83 (m, 2H), 7.53 (dd, J = 22.4, 8.9 Hz, 1H), 7.43 – 7.21 (m, 6H), 7.05 (s, 1H), 5.19 – 4.87 (m, 2H), 4.43 – 4.06 (m, 4H), 3.59 – 3.32 (m, 2H), 2.18 (ddd, J = 17.3, 11.3, 7.1 Hz, 3H), 1.97 – 1.46 (m, 8H), 1.38 (d, J = 3.0 Hz, 9H), 0.97 – 0.68 (m, 12H). ¹³C NMR (75 MHz, DMSO-d₆) δ 173.0, 172.54, 172.50, 172.40, 172.19, 171.11, 137.44, 128.82, 128.62, 128.22, 127.90, 127.28, 80.09, 66.37, 66.16, 60.29, 59.36, 57.68, 57.60, 52.20, 52.07, 51.65, 51.42, 47.57, 47.00, 41.05, 40.72, 31.62, 31.06, 30.99, 30.45, 28.20, 27.75, 27.60, 24.65, 24.57, 24.33, 23.39, 23.33, 22.02, 19.68, 18.11. HRMS (ESI) calcd for C₃₃H₅₁N₅O₈Na 668.3630 (M + Na)⁺, found 668.3630.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-4-methyl-2-((S)-pyrrolidine-2-carboxamido)pentanamido)-5-oxopentanoate (12).

To a solution of compound 11 (1.1 g, 0.85 mmol) in 100 mL of MeOH, 10% Pd/C (100 mg) was added. Under H₂, the mixture was allowed to stir at room temperature overnight. The solution was filtered and the filtrate was concentrated to get the crude product. The residue was purified by silica gel column (CH₂Cl₂/MeOH = 50/1 to 25/1) to obtain compound 12 (793 mg, 91%) as a white solid. ¹H NMR (300 MHz, Methanol-d₄) δ 4.54 – 4.39 (m, 2H), 4.23 (d, J = 6.7 Hz, 1H), 3.69 (dd, J = 8.7, 5.4 Hz, 1H), 3.33 (q, J = 1.6 Hz, 1H), 2.96 (tdd, J = 10.4, 6.3, 3.9 Hz, 2H), 2.34 (dt, J = 8.6, 6.5 Hz, 2H), 2.21 – 2.01 (m, 3H), 1.98 – 1.59 (m, 7H), 1.46 (s, 9H), 1.06 – 0.88 (m, 12H). ¹³C NMR (75 MHz, Methanol-d₄) δ 175.81, 174.44, 173.34, 172.58, 171.94, 80.40, 60.11, 58.28, 52.46, 51.52, 47.33, 47.05, 46.76, 46.67, 40.82, 31.19, 30.70, 30.60, 26.98, 26.86, 25.67, 24.65, 22.01, 20.72, 18.38, 17.04. HRMS (ESI) calcd for C₂₅H₄₆N₅O₆ 512.3443 (M + H)⁺, found 512.3440.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-2-((S)-1-benzoylpyrrolidine-2-carboxamido)-4-methylpentanamido)-5-oxopentanoate (13a).

A solution of compound 12 (102 mg, 0.2 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Then Et₃N (65 mg, 0.5 mmol) and benzoyl chloride (42 mg, 0.3 mmol) were added. The mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and the solution washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain compound 13a (104 mg, 85%) as a white solid. ¹H NMR (300 MHz, Methanol-d₄) δ 7.73 – 7.31 (m, 5H), 4.66 – 4.34 (m, 3H), 4.23 (d, J = 6.5 Hz, 1H), 3.92 – 3.48 (m, 3H), 2.43 – 2.27 (m, 3H), 2.21 – 1.58 (m, 9H), 1.49 – 1.35 (m, 13H), 1.02 – 0.83 (m, 12H). ¹³C NMR (75 MHz, Methanol-d₄) δ 173.53, 172.48, 172.07, 130.14, 128.05, 126.92, 126.40, 80.35, 60.72, 58.39, 52.66, 52.24, 50.31, 47.31, 47.03, 46.74, 40.18, 31.18, 30.53, 29.59, 26.96, 26.83, 25.01, 24.51, 22.05, 20.64, 18.37, 17.33, 17.02, 15.89. HRMS (ESI) calcd for C₃₂H₅₀N₅O₇ 616.3705 (M + H)⁺, found 616.3702.

tert-Butyl (S)-4-((S)-2-((S)-1-(((3R,5R,7R)-adamantan-1-yl)carbamoyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (13b).

A solution of compound 12 (51 mg, 0.1 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Followed by adding Et₃N (33 mg, 0.25 mmol) and 1-adamantyl isocyanate (18 mg, 0.1 mmol), the mixture solution was stirring at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and the solution washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain compound 13b (57 mg, 84%) as a white solid. ¹H NMR (300 MHz, Methanol-d₄) δ 4.44 (dd, J = 9.2, 5.4 Hz, 1H), 4.37 – 4.25 (m, 2H), 4.22 (d, J = 6.6 Hz, 1H), 3.51 (d, J = 7.1 Hz, 1H), 2.34 (td, J = 7.6, 7.1, 2.5 Hz, 2H), 2.25 – 2.12 (m, 3H), 2.11 – 1.93 (m, 14H), 1.78 – 1.60 (m, 9H), 1.46 (s, 9H), 1.08 – 0.85 (m, 12H). ¹³C NMR (75 MHz, Methanol-d₄) δ 174.94, 173.76, 172.27, 80.38, 60.55, 58.63, 52.96, 52.56, 51.18, 46.29, 41.79, 40.07, 36.16, 31.26, 30.41, 29.66, 29.54, 26.95, 26.73, 24.60, 24.39, 22.01, 20.57, 18.34, 17.09. HRMS (ESI) calcd for C₃₆H₆₀N₆O₇Na 711.4416 (M + Na)⁺, found 711.4415.

tert-Butyl (S)-4-((S)-2-((S)-1-((3S,5S,7S)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (13c).

A solution of compound 12 (51 mg, 0.1 mmol) and 1-adamantanecarboxylic acid (18 mg, 0.1 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Then, HOBt (15 mg, 0.1 mmol), HBTU (76 mg, 0.2 mmol) and DIPEA (32 μL, 0.2 mmol) were added to the solution at 0 °C. The mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and the mixture washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 13c (49 mg, 73%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.84 (d, J = 6.2 Hz, 1H), 7.10 (d, J = 9.0 Hz, 1H), 6.93 – 6.74 (m, 2H), 5.33 (s, 1H), 4.62 (s, 1H), 4.43 (dd, J = 9.0, 5.3 Hz, 1H), 4.28 (q, J = 6.3, 5.7 Hz, 1H), 4.16 – 4.04 (m, 1H), 3.97 – 3.65 (m, 3H), 3.18 (dd, J = 7.5, 4.4 Hz, 1H), 2.45 (d, J = 3.6 Hz, 3H), 2.24 – 2.00 (m, 11H), 1.83 – 1.66 (m, 7H), 1.52 – 1.37 (m, 15H), 1.11 – 0.80 (m, 12H). ¹³C NMR (75 MHz, Chloroform-d) δ 173.95, 171.68, 77.19, 62.79, 58.59, 55.18, 55.02, 54.04, 48.78, 42.18, 40.21, 38.18, 36.41, 32.34, 29.08, 28.13, 28.09, 26.56, 26.31, 26.19, 25.19, 23.19, 21.42, 19.47, 18.65, 17.48, 17.30. HRMS (ESI) calcd for C₃₆H₅₉N₅O₇Na 696.4307 (M + Na)⁺, found 696.4302.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-2-((S)-1-decanoylpyrrolidine-2-carboxamido)-4-methylpentanamido)-5-oxopentanoate (13d).

A solution of compound 12 (51 mg, 0.1 mmol) and capric acid (17 mg, 0.1 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Then, HOBt (14 mg, 0.1 mmol), HBTU (76 mg, 0.2 mmol) and DIPEA (32 μL, 0.2 mmol) were added to the solution at 0 °C. Then removed the ice bath, and the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 13d (48 mg, 72%) as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ 8.33 – 7.77 (m, 2H), 7.65 – 7.24 (m, 2H), 7.06 (s, 1H), 4.44 – 4.04 (m, 4H), 3.69 – 3.40 (m, 3H), 2.32 – 1.60 (m, 11H), 1.39 (s, 12H), 1.26 (dd, J = 11.1, 4.5 Hz, 14H), 0.84 (m, 14H). ¹³C NMR (75 MHz, DMSO-d₆) δ 173.06, 172.57, 172.40, 172.14, 171.21, 80.07, 60.13, 59.70, 57.82, 53.94, 52.36, 51.80, 42.19, 40.71, 40.40, 39.57, 34.25, 33.88, 32.15, 31.74, 31.67, 30.87, 29.66, 29.46, 29.39, 29.28, 29.20, 29.12, 28.20, 27.67, 27.38, 24.84, 24.78, 24.71, 24.63, 23.50, 23.44, 22.75, 22.54, 21.89, 19.68, 18.51, 18.11, 17.19, 14.39, 12.81. HRMS (ESI) calcd for C₃₅H₆₄N₅O₇ 666.4800 (M + H)⁺, found 666.4796.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-2-((S)-1-(cyclohexanecarbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-oxopentanoate (13e).

A solution of compound 12 (51 mg, 0.1 mmol) and cyclohexanecarboxylic acid (13 mg, 0.1 mmol in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. Followed by adding HOBt (14 mg, 0.1 mmol), HBTU (76 mg, 0.2 mmol) and DIPEA (32 μL, 0.2 mmol) to the solution at 0 °C. Then ice bath was removed, and the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 13e (51 mg, 82%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 8.00 (s, 1H), 7.39 (s, 1H), 7.18 (s, 1H), 6.89 (s, 1H), 5.36 (s, 1H), 4.59 (s, 1H), 4.40 (s, 1H), 4.26 (d, J = 8.6 Hz, 1H), 4.07 (s, 1H), 3.64 (d, J = 33.5 Hz, 3H), 3.14 (d, J = 7.9 Hz, 2H), 2.58 – 2.32 (m, 4H), 2.15 (t, J = 31.3 Hz, 7H), 1.74 (s, 6H), 1.50 (d, J = 8.4 Hz, 12H), 1.15 – 0.77 (m, 12H). ¹³C NMR (75 MHz, Chloroform-d) δ 174.08, 173.65, 173.09, 77.19, 60.34, 58.72, 55.10, 54.36, 54.17, 47.41, 42.78, 42.50, 40.04, 32.26, 29.27, 29.09, 28.43, 28.07, 27.53, 25.97, 25.67, 25.60, 25.49, 25.14, 24.95, 23.13, 21.17, 19.43, 18.62, 17.47, 17.33, 12.18. HRMS (ESI) calcd for C₃₂H₅₅N₅O₇Na 644.3994 (M + Na)⁺, found 644.3989.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-2-((S)-1-(4-fluorobenzoyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-oxopentanoate (13f).

A solution of compound 12 (51 mg, 0.1 mmol) and 4-fluorobenzoic acid (14 mg, 0.1 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath, followed by adding HOBt (14 mg, 0.1 mmol), HBTU (76 mg, 0.2 mmol) and DIPEA (32 μL, 0.2 mmol) to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 13f (52 mg, 83%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.20 – 7.81 (m, 2H), 7.69 – 7.47 (m, 2H), 7.45 – 7.22 (m, 3H), 7.20 – 6.90 (m, 2H), 4.52 – 3.99 (m, 4H), 3.69 – 3.46 (m, 3H), 2.32 – 2.07 (m, 3H), 2.00 – 1.65 (m, 6H), 1.56 – 1.21 (m, 11H), 1.04 – 0.51 (m, 12H). ¹³C NMR (75 MHz, DMSO-d₆) δ 173.04, 172.17, 171.97, 171.13, 130.36, 130.24, 129.70, 115.69, 115.42, 80.09, 61.68, 60.59, 57.71, 51.68, 50.36, 40.69, 40.40, 39.84, 39.28, 31.61, 31.06, 30.97, 29.89, 28.19, 27.59, 25.34, 24.66, 24.30, 23.46, 22.74, 22.09, 21.68, 19.68, 18.12. HRMS (ESI) calcd for C₃₂H₄₈FN₅O₈Na 656.3430 (M + Na)⁺, found 656.3424.

tert-Butyl (S)-5-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-2-((S)-1-((4-fluorophenyl)sulfonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-oxopentanoate (13g).

A solution of compound 12 (51 mg, 0.1 mmol) in 5 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath, followed by adding regents Et₃N (33 mg, 0.25 mmol) and 4-fluorobenzenesulfonyl chloride (19 mg, 0.1 mmol) to the solution. The mixture was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CHCl₃/CH₃OH = 50/1 to 20/1) to obtain compound 13g (47 mg, 70%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.26 – 7.83 (m, 4H), 7.65 – 7.29 (m, 4H), 7.05 (s, 1H), 4.44 – 4.02 (m, 4H), 3.44 – 3.36 (m, 2H), 3.14 (dd, J = 17.7, 9.1 Hz, 1H), 2.21 (q, J = 8.4, 7.7 Hz, 2H), 1.95 – 1.62 (m, 7H), 1.53 – 1.47 (m, 2H), 1.37 (s, 9H), 0.85 (dt, J = 18.9, 7.0 Hz, 12H). ¹³C NMR (75 MHz, DMSO-d₆) δ 173.04, 172.34, 172.16, 171.36, 171.09, 130.92, 130.80, 117.06, 116.76, 80.08, 61.35, 57.61, 52.23, 51.32, 49.49, 41.14, 31.68, 31.22, 31.05, 28.19, 27.54, 24.62, 24.55, 23.53, 22.08, 19.68, 18.12. HRMS (ESI) calcd for C₃₁H₄₈FN₅O₈SNa 692.3100 (M + Na)⁺, found 692.3094.

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (15).

A solution of Fmoc-Glu(O^tBu)-OH (6) (1.3 g, 3 mmol) and H-Val-OMe•HCl (14) (504 mg, 3 mmol) in 20 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath, followed by adding regents HOBt (405 mg, 3 mmol), HBTU (2.3 g, 6 mmol) and DIPEA (2 mL, 12 mmol) to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 15 (1.5 g, 89%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.4 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.46 – 7.38 (m, 2H), 7.33 (td, J = 7.4, 1.2 Hz, 2H), 6.98 (d, J = 8.5 Hz, 1H), 5.77 (d, J = 7.7 Hz, 1H), 4.53 (dd, J = 8.6, 4.8 Hz, 1H), 4.45 – 4.37 (m, 2H), 4.25 (d, J = 7.1 Hz, 2H), 3.75 (s, 3H), 2.46 (t, J = 6.6 Hz, 2H), 2.29 – 1.94 (m, 3H), 1.49 (s, 9H), 0.96 (dd, J = 8.9, 6.9 Hz, 6H).

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (17).

Compound 15 (1.08 g, 2 mmol) was added to a mixture solution of HNEt₂ (2 mL) and MeCN (8 mL) at room temperature and the solution was stirred at the same temperature for 1 h. After the reaction finished (monitored by TLC), the solvent was removed under vacuum to generate compound 16. Then, compound 10 (724, 2 mmol) was added to the CH₂Cl₂ (20 mL) solution of compound 16, and the solution was cooled to 0 °C with an ice bath. HOBt (270 mg, 2 mmol), HBTU (1.5 g, 4 mmol) and DIPEA (1.3 mL, 8 mmol) were added to the solution at the same temperature. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain compound 17 (977 mg, 74%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.36 (s, 6H), 7.10 (d, J = 8.6 Hz, 1H), 6.90 – 6.78 (m, 1H), 5.17 (s, 2H), 4.54 – 4.31 (m, 4H), 3.73 (s, 3H), 3.54 (d, J = 8.3 Hz, 2H), 2.39 (dt, J = 11.0, 7.3 Hz, 2H), 2.20 (pd, J = 6.9, 5.3 Hz, 3H), 2.04 – 1.89 (m, 3H), 1.77 – 1.44 (m, 13H), 0.92 (ddd, J = 13.8, 8.3, 5.4 Hz, 12H). ¹³C NMR (75 MHz, Chloroform-d) δ 173.07, 171.94, 171.02, 136.23, 128.53, 128.17, 127.86, 80.86, 67.54, 60.93, 57.51, 52.38, 52.03, 47.15, 40.45, 31.97, 30.79, 28.86, 28.07, 27.26, 25.02, 24.69, 22.94, 21.58, 18.99, 17.81. HRMS (ESI) calcd for C₃₄H₅₂N₄O₉Na 683.3627 (M + Na)⁺, found 683.3626.

((S)-2-((S)-2-((S)-1-((Benzyloxy)carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(tert-butoxy)-5-oxopentanoyl)-L-valine (18).

To a solution of 17 (660 mg, 1 mmol) in 8 mL of MeOH at 0 °C, LiOH∙H₂O (84 mg, 2 mmol) in 2 mL of H₂O was added. The solution was allowed to stir at room temperature overnight, and then acidified to pH = 3 by 1 N NaHSO₄. The mixture was extracted by CH₂Cl₂ (3 × 15 mL) and the organic layer was dried over Na₂SO₄. After concentration, compound 18 (450 mg, 67%) was obtained without further purification as a white foam. ¹H NMR (300 MHz, Chloroform-d) δ 7.36 (q, J = 6.4, 5.3 Hz, 7H), 7.08 (d, J = 7.2 Hz, 1H), 5.37 – 4.98 (m, 3H), 4.62 – 4.29 (m, 4H), 3.55 (s, 2H), 2.47 – 1.86 (m, 9H), 1.44 (s, 11H), 0.94 (ddd, J = 25.5, 8.4, 5.2 Hz, 12H).

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-3-methyl-1-morpholino-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19a).

To a solution of 18 (69 mg, 0.1 mmol) in CH₂Cl₂ (5 mL) at 0 °C, morpholine (0.015 mL, 0.2 mmol), HOBt (14 mg, 40.1 mmol), HBTU (57 mg, 0.15 mmol) and DIPEA (0.07 mL, 0.4 mmol) were added to the solution at 0 °C. Then the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain compound 19a (57 mg, 80%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.34 (s, 5H), 7.27 – 7.17 (m, 1H), 7.12 – 6.64 (m, 1H), 5.16 (s, 2H), 4.67 (t, J = 8.0 Hz, 1H), 4.49 (td, J = 8.2, 5.0 Hz, 1H), 4.35 (q, J = 7.6, 5.5 Hz, 2H), 3.87 – 3.48 (m, 10H), 3.35 (d, J = 4.6 Hz, 1H), 2.98 (d, J = 8.5 Hz, 2H), 2.49 – 1.82 (m, 9H), 1.77 – 1.49 (m, 2H), 1.42 (s, 9H), 1.01 – 0.77 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.61, 172.28, 172.01, 170.84, 169.76, 166.61, 156.14, 136.29, 128.50, 128.15, 127.81, 80.72, 67.48, 66.83, 66.72, 65.98, 60.90, 53.50, 52.76, 52.42, 50.61, 48.79, 47.12, 46.30, 42.42, 40.37, 38.57, 31.92, 31.00, 28.99, 28.06, 27.32, 24.98, 24.66, 22.90, 21.67, 19.64, 17.64. HRMS (ESI) calcd for C₃₇H₅₇N₅O₉Na 738.4048 (M + Na)⁺, found 738.4042.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-(diethylamino)-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19b).

Compound 19b (46 mg, 65%) was prepared by a similar synthetic process to produce compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.31 (m, 5H), 7.21 – 7.05 (m, 3H), 5.16 (s, 2H), 4.81 – 4.19 (m, 4H), 3.48 (dq, J = 32.6, 6.2, 5.5 Hz, 4H), 3.19 (dd, J = 13.6, 7.0 Hz, 1H), 2.40 – 1.85 (m, 10H), 1.57 (d, J = 31.5 Hz, 2H), 1.42 (d, J = 2.0 Hz, 9H), 1.26 – 1.21 (m, 4H), 1.10 (t, J = 7.0 Hz, 3H), 1.02 – 0.76 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.44, 171.90, 170.67, 170.51, 156.00, 136.42, 131.99, 128.54, 128.48, 128.45, 128.06, 127.80, 80.60, 67.36, 60.63, 53.87, 52.61, 47.08, 42.07, 40.68, 40.40, 31.71, 29.66, 28.92, 28.05, 24.88, 22.89, 21.80, 19.58, 19.49, 17.80, 14.62, 14.57, 12.87, 11.74. HRMS (ESI) calcd for C₃₇H₆₀N₅O₈ 702.4436 (M + H)⁺, found 702.4434.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-3-methyl-1-(methylamino)-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19c).

Compound 19c (56 mg, 86%) was prepared by a similar synthetic process to produce compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.87 (d, J = 6.2 Hz, 1H), 7.38 (s, 5H), 7.02 (d, J = 5.2 Hz, 1H), 6.91 (d, J = 9.3 Hz, 1H), 6.74 (s, 1H), 5.20 (s, 2H), 4.49 – 4.12 (m, 4H), 3.57 (d, J = 7.2 Hz, 2H), 2.80 (d, J = 4.7 Hz, 3H), 2.53 – 2.31 (m, 3H), 2.25 – 2.05 (m, 4H), 2.02 – 1.90 (m, 2H), 1.67 (d, J = 10.8 Hz, 4H), 1.45 (s, 9H), 0.92 (dd, J = 17.1, 6.0 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.44, 173.18, 172.69, 171.55, 171.37, 156.59, 136.07, 128.61, 128.35, 127.82, 81.15, 67.73, 60.93, 58.68, 54.77, 53.71, 47.18, 40.22, 32.32, 29.15, 28.61, 28.08, 26.23, 25.10, 24.72, 22.96, 21.41, 19.44, 17.28. HRMS (ESI) calcd for C₃₄H₅₄N₅O₈ 660.3972 (M + H)⁺, found 660.3970.

Benzyl (S)-2-(((S)-1-(((S)-1-(((S)-1-(benzylamino)-3-methyl-1-oxobutan-2-yl)amino)-5-(tert-butoxy)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19d).

Compound 19d (57 mg, 75%) was prepared by a similar synthetic process to produce compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.87 (d, J = 6.4 Hz, 1H), 7.37 (s, 5H), 7.28 (s, 5H), 7.22 – 7.16 (m, 2H), 7.09 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 5.7 Hz, 1H), 5.18 (s, 2H), 4.59 – 4.27 (m, 6H), 3.52 (t, J = 7.2 Hz, 2H), 2.40 (s, 4H), 2.20 – 1.93 (m, 6H), 1.60 (s, 2H), 1.44 (s, 9H), 0.92 (dd, J = 17.3, 6.1 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.21, 172.90, 172.53, 172.09, 171.49, 171.02, 156.41, 138.66, 136.14, 128.57, 128.26, 127.83, 127.64, 126.84, 81.01, 67.65, 60.92, 58.65, 54.48, 53.40, 47.12, 43.26, 40.35, 39.84, 32.24, 29.59, 28.75, 28.08, 26.56, 25.01, 24.69, 22.89, 21.56, 19.53, 19.46, 17.52. HRMS (ESI) calcd for C₄₀H₅₈N₅O₈ 736.4285 (M + H)⁺, found 736.4280.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-3-methyl-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19e).

Compound 19e (53 mg, 76%) was prepared by a similar synthetic process to yield compound 19e as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.34 (s, 5H), 7.21 (d, J = 8.6 Hz, 1H), 7.15 (t, J = 4.3 Hz, 1H), 6.98 (s, 1H), 5.16 (s, 2H), 4.44 (dt, J = 41.0, 6.4 Hz, 4H), 3.73 – 3.38 (m, 7H), 2.29 (d, J = 7.9 Hz, 2H), 2.08 – 1.83 (m, 10H), 1.67 – 1.52 (m, 3H), 1.41 (s, 9H), 0.89 (dt, J = 11.9, 5.0 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.54, 172.28, 171.99, 170.83, 169.72, 156.04, 136.38, 128.46, 128.06, 127.80, 80.63, 67.37, 60.73, 56.03, 52.67, 49.44, 47.08, 46.72, 45.84, 40.12, 31.09, 29.65, 29.01, 28.05, 27.60, 26.01, 25.07, 24.91, 24.16, 22.89, 19.45, 17.87. HRMS (ESI) calcd for C₃₇H₅₈N₅O₈ 700.4285 (M + H)⁺, found 700.4284.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-(3-hydroxyazetidin-1-yl)-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19f).

Compound 19f (46 mg, 66%) was prepared by a similar synthetic process to yield compound 19e as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.51 (s, 1H), 7.35 (s, 5H), 7.15 (d, J = 8.7 Hz, 1H), 6.87 (s, 1H), 5.17 (s, 2H), 4.64 – 3.99 (m, 9H), 3.55 (q, J = 8.1, 6.8 Hz, 2H), 2.45 – 1.81 (m, 10H), 1.75 – 1.50 (m, 3H), 1.43 (s, 9H), 0.91 (d, J = 6.5 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.75, 172.46, 171.26, 170.74, 156.43, 136.44, 128.53, 128.19, 127.80, 80.92, 67.56, 61.25, 60.37, 58.26, 57.61, 54.34, 53.68, 52.92, 47.20, 40.29, 31.94, 28.07, 27.26, 25.04, 24.67, 22.91, 21.63, 19.19, 18.14. HRMS (ESI) calcd for C₃₆H₅₆N₅O₉ 702.4078 (M + H)⁺, found 702.4074.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-(cyclohexylamino)-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19g).

Compound 19g (54 mg, 74%) was prepared by a similar synthetic process to produce compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.85 (d, J = 6.4 Hz, 1H), 7.34 (d, J = 14.5 Hz, 5H), 7.04 (d, J = 5.4 Hz, 1H), 7.00 (s, 1H), 6.64 (s, 1H), 5.18 (s, 2H), 4.49 – 4.11 (m, 4H), 3.72 (s, 1H), 3.55 (q, J = 7.4, 6.5 Hz, 2H), 2.38 (d, J = 7.1 Hz, 2H), 2.13 (dq, J = 16.0, 8.6, 7.1 Hz, 4H), 1.90 (dt, J = 24.2, 9.1 Hz, 6H), 1.76 – 1.55 (m, 6H), 1.43 (s, 10H), 1.25 – 1.15 (m, 3H), 0.90 (dd, J = 15.6, 7.7 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.12, 172.54, 171.32, 169.82, 156.41, 136.16, 128.55, 128.28, 127.84, 80.93, 67.65, 61.01, 58.64, 54.85, 54.35, 53.38, 48.34, 47.16, 40.41, 39.93, 33.39, 32.90, 32.70, 32.25, 29.57, 28.88, 28.07, 26.64, 25.57, 25.04, 25.00, 24.72, 22.89, 21.58, 19.40, 17.45. HRMS (ESI) calcd for C₃₉H₆₂N₅O₈ 728.4593 (M + H)⁺, found 728.4592.

Benzyl (S)-2-(((S)-1-(((S)-1-(((S)-1-(((3R,5R,7R)-adamantan-1-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-(tert-butoxy)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19h).

Compound 19h (55 mg, 71%) was obtained by a similar synthetic process to prepare compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.69 (d, J = 5.3 Hz, 1H), 7.37 (s, 5H), 6.95 (d, J = 13.6 Hz, 2H), 6.08 (s, 1H), 5.18 (s, 2H), 4.60 – 4.19 (m, 4H), 3.54 (s, 2H), 2.38 (s, 3H), 2.13 (d, J = 7.6 Hz, 3H), 2.09 – 1.83 (m, 16H), 1.66 (s, 9H), 1.44 (s, 9H), 0.99 – 0.83 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.09, 172.35, 171.93, 171.14, 169.84, 169.78, 156.38, 136.19, 132.14, 132.01, 128.55, 128.51, 128.27, 127.84, 80.88, 80.67, 67.62, 60.93, 59.13, 53.97, 53.16, 51.99, 47.15, 41.47, 41.26, 40.51, 36.40, 32.22, 29.70, 29.47, 29.42, 28.79, 28.08, 26.77, 25.03, 24.72, 23.09, 22.91, 21.59, 19.46, 19.37, 18.03, 17.58. HRMS (ESI) calcd for C₄₃H₆₆N₅O₈ 780.4911 (M + H)⁺, found 780.4904.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-3-methyl-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19i).

Compound 19i (44 mg, 60%) was obtained by a similar synthetic process to prepare compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.34 (s, 5H), 7.18 (d, J = 8.3 Hz, 1H), 7.08 – 6.60 (m, 1H), 5.16 (s, 2H), 4.74 (t, J = 7.7 Hz, 1H), 4.50 (q, J = 8.1, 5.5 Hz, 1H), 4.36 (q, J = 8.2, 5.5 Hz, 2H), 3.74 – 3.41 (m, 6H), 2.39 (t, J = 9.6 Hz, 5H), 2.30 (s, 4H), 2.24 – 1.81 (m, 7H), 1.57 (d, J = 37.3 Hz, 3H), 1.42 (s, 9H), 1.01 – 0.70 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.57, 172.14, 171.91, 170.75, 169.51, 156.12, 136.34, 128.48, 128.10, 127.81, 80.68, 67.42, 60.78, 55.14, 54.68, 53.45, 52.71, 52.31, 47.09, 45.91, 45.68, 41.99, 40.56, 31.87, 31.22, 29.66, 28.91, 28.07, 27.49, 24.94, 24.66, 22.89, 21.75, 19.76, 19.69, 17.45. HRMS (ESI) calcd for C₃₈H₆₁N₆O₈ 729.4551 (M + H)⁺, found 729.4544.

Benzyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-((2-hydroxyethyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19j).

Compound 19j (22 mg, 30%) was produced by a similar synthetic process to prepare compound 19a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 8.07 (d, J = 5.7 Hz, 1H), 7.38 (s, 5H), 7.17 (d, J = 4.4 Hz, 1H), 7.02 – 6.88 (m, 2H), 5.20 (s, 2H), 4.60 – 3.90 (m, 5H), 3.68 (d, J = 11.4 Hz, 2H), 3.62 – 3.41 (m, 3H), 3.35 (d, J = 5.8 Hz, 1H), 2.60 – 2.34 (m, 3H), 2.14 (dq, J = 8.0, 5.0 Hz, 3H), 2.05 – 1.87 (m, 3H), 1.65 (dd, J = 9.4, 4.4 Hz, 2H), 1.45 (s, 9H), 0.97 – 0.81 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 174.04, 173.55, 173.47, 172.76, 171.43, 171.36, 156.71, 135.98, 128.63, 128.61, 128.41, 127.83, 81.32, 67.83, 61.73, 60.97, 58.40, 55.51, 54.31, 47.21, 42.84, 40.25, 32.42, 29.68, 28.94, 28.56, 28.09, 25.93, 25.07, 24.73, 22.82, 21.55, 19.54, 19.49, 17.34. HRMS (ESI) calcd for C₃₅H₅₆N₅O₉ 690.4078 (M + H)⁺, found 690.4069.

Benzyl (S)-2-(((S)-1-(((S)-1-(((S)-1-(allyloxy)-3-methyl-1-oxobutan-2-yl)amino)-5-(tert-butoxy)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (19k).

To a solution of 18 (69 mg, 0.1 mmol) in 2 mL of dry DMF, NaHCO₃ (13 mg, 0.15 mmol), allyl bromide (24 mg, 0.2 mmol) and TBAI (7 mg, 0.02 mmol) were added. The mixture was allowed to stir at room temperature overnight. After the reaction completed (detected by TLC), diluted with 10 mL of water. And then, the mixture was extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with brine and dried over anhydrous Na₂SO₄. Then the solution was concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain 23a (52 mg, 76%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.36 (s, 5H), 7.18 – 6.95 (m, 2H), 6.83 (d, J = 7.4 Hz, 1H), 5.92 (ddt, J = 16.5, 11.0, 5.7 Hz, 1H), 5.34 (dq, J = 17.2, 1.5 Hz, 1H), 5.28 – 5.21 (m, 1H), 5.17 (s, 2H), 4.63 (td, J = 5.1, 4.4, 1.5 Hz, 2H), 4.53 – 4.31 (m, 4H), 3.53 (dd, J = 12.6, 6.7 Hz, 2H), 2.50 – 2.34 (m, 2H), 2.30 – 2.06 (m, 4H), 2.05 – 1.83 (m, 4H), 1.81 – 1.54 (m, 2H), 1.44 (s, 9H), 0.98 – 0.82 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.09, 171.94, 171.11, 171.00, 166.59, 156.32, 136.25, 131.76, 128.53, 128.17, 127.86, 118.67, 80.84, 67.52, 65.66, 60.88, 57.49, 52.63, 52.32, 47.13, 40.50, 32.00, 30.82, 28.79, 28.07, 27.32, 25.00, 24.69, 22.95, 21.59, 19.03, 17.73. HRMS (ESI) calcd for C₃₆H₅₅N₄O₉ 687.3969 (M + H)⁺, found 687.3962.

tert-Butyl (S)-5-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-4-((S)-4-methyl-2-((S)-pyrrolidine-2-carboxamido)pentanamido)-5-oxopentanoate (20).

To a solution of compound 17 (1.0 g) in 100 mL of MeOH, 10% Pd/C (100 mg) was added. Under H₂, the mixture was allowed to stir at room temperature for 12 hrs. The solution was filtrated and the filtrate was concentrated to get the crude product. The residue was purified by column chromatography (CH₂Cl₂/MeOH= 20/1 to 15/1) to obtain compound 20 (733 mg, 92%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 8.00 (d, J = 8.3 Hz, 1H), 7.32 – 7.24 (m, 1H), 7.19 (d, J = 8.5 Hz, 1H), 4.55 – 4.36 (m, 3H), 3.82 (dd, J = 9.1, 5.3 Hz, 1H), 3.72 (s, 3H), 3.55 – 3.27 (m, 1H), 3.06 – 2.91 (m, 2H), 2.82 (s, 2H), 2.39 (q, J = 6.9 Hz, 2H), 2.21 – 2.03 (m, 3H), 1.90 (tt, J = 12.7, 6.4 Hz, 2H), 1.77 – 1.54 (m, 6H), 1.44 (s, 9H), 1.25 (t, J = 7.1 Hz, 1H), 1.10 (t, J = 7.2 Hz, 1H), 0.95 – 0.88 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 175.26, 173.22, 172.33, 171.98, 171.12, 80.98, 60.38, 57.53, 52.48, 52.04, 51.47, 47.22, 40.84, 31.73, 30.73, 30.67, 28.05, 27.46, 26.05, 24.93, 22.95, 21.78, 18.97, 17.75. HRMS (ESI) calcd for C₂₆H₄₇N₄O₇ 527.3445 (M + H)⁺, found 527.3434.

tert-Butyl (S)-4-((S)-2-((S)-1-((3S,5S,7S)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (21a).

A solution of compound 20 (53 mg, 0.1 mmol) and 1-adamantanecarboxylic acid (18 mg, 0.1 mmol) in CH₂Cl₂ (5 mL) was cooled to 0 °C with an ice bath. HOBt (15 mg, 0.1 mmol), HBTU (76 mg, 0.2 mmol) and DIPEA (32 μL, 0.2 mmol) were added to the solution at the same temperature. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 20 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain compound 21a (46 mg, 67%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.26 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 7.0 Hz, 1H), 4.70 – 4.60 (m, 1H), 4.48 (td, J = 8.0, 5.3 Hz, 2H), 4.32 (ddd, J = 10.7, 6.9, 4.4 Hz, 1H), 3.74 (s, 5H), 2.41 (dt, J = 10.8, 7.4 Hz, 2H), 2.27 – 2.12 (m, 3H), 2.10 – 1.94 (m, 12H), 1.82 – 1.61 (m, 10H), 1.46 (s, 9H), 1.03 – 0.85 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.64, 172.75, 172.30, 172.02, 171.86, 171.10, 80.64, 62.28, 57.56, 52.60, 52.41, 51.91, 48.55, 42.01, 40.46, 38.14, 38.12, 36.58, 36.46, 32.01, 30.70, 28.30, 28.18, 28.03, 27.32, 24.94, 23.15, 21.50, 18.99, 17.94. HRMS (ESI) calcd for C₃₇H₆₁N₄O₈ 689.4489 (M + H)⁺, found 689.4482.

(9H-Fluoren-9-yl)methyl (S)-2-(((S)-1-(((S)-5-(tert-butoxy)-1-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-1,5-dioxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (21b).

Compound 21b (55 mg, 73%) was obtained by a similar synthetic process to prepare compound 13a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.28 (s, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 7.4 Hz, 1H), 4.51 – 4.22 (m, 7H), 3.73 (s, 3H), 3.60 – 3.41 (m, 2H), 3.34 – 3.19 (m, 1H), 2.40 (dq, J = 16.0, 7.0, 5.8 Hz, 2H), 2.27 – 2.12 (m, 3H), 1.96 (s, 3H), 1.81 – 1.52 (m, 3H), 1.43 (s, 9H), 0.91 (ddd, J = 13.9, 6.5, 4.0 Hz, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.15, 171.90, 170.98, 156.35, 143.73, 141.33, 141.26, 127.78, 127.63, 127.08, 125.17, 124.95, 120.00, 119.86, 80.88, 67.81, 60.82, 57.48, 52.62, 52.31, 52.02, 47.20, 47.05, 41.75, 41.28, 40.49, 31.96, 30.78, 28.05, 27.29, 25.02, 24.74, 23.45, 22.94, 21.55, 18.99, 17.78, 14.19, 13.81. HRMS (ESI) calcd for C₄₁H₅₇N₄O₉ 749.4126 (M + H)⁺, found 749.4115.

tert-Butyl (S)-4-((S)-2-((S)-1-acetylpyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (21c).

Compound 21c (45 mg, 78%) was produced by a similar synthetic process to prepare compound 13a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.31 – 7.23 (m, 2H), 7.17 (d, J = 8.5 Hz, 1H), 4.59 – 4.41 (m, 3H), 4.32 (ddd, J = 9.3, 7.0, 4.7 Hz, 1H), 3.71 (s, 3H), 3.57 (q, J = 5.5, 3.5 Hz, 1H), 3.46 (q, J = 9.4, 8.6 Hz, 1H), 2.47 – 2.16 (m, 5H), 2.10 (s, 3H), 2.05 – 1.92 (m, 4H), 1.71 (d, J = 12.9 Hz, 1H), 1.59 (dt, J = 14.4, 7.6 Hz, 2H), 1.44 (s, 9H), 0.95 – 0.85 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 173.18, 172.04, 171.92, 171.70, 171.10, 80.85, 59.86, 57.49, 52.60, 52.48, 51.99, 48.29, 40.42, 31.87, 30.76, 28.07, 27.83, 27.38, 25.07, 24.97, 22.94, 22.43, 21.60, 18.98, 17.79. HRMS (ESI) calcd for C₂₈H₄₉N₄O₈ 569.3550 (M + H)⁺, found 569.35540.

((S)-2-((S)-2-((S)-1-((3S,5S,7S)-Adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(tert-butoxy)-5-oxopentanoyl)-L-valine (22).

Compound 22 (1.6 g, 80%) was obtained by a similar synthetic process to prepare compound 18 from 21a as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.53 (d, J = 7.7 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 6.9 Hz, 1H), 4.62 (d, J = 6.9 Hz, 1H), 4.50 (q, J = 7.3 Hz, 1H), 4.43 – 4.29 (m, 2H), 3.90 – 3.77 (m, 2H), 2.44 – 2.22 (m, 3H), 2.20 – 1.91 (m, 16H), 1.81 – 1.53 (m, 10H), 1.45 (s, 9H), 1.02 – 0.88 (m, 12H).

tert-Butyl (S)-4-((S)-2-((S)-1-((3S,5S,7S)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-(allyloxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (23a).

To a solution of 22 (68 mg, 0.1 mmol) in 2 mL of dry DMF, NaHCO₃ (13 mg, 0.15 mmol), allyl bromide (24 mg, 0.2 mmol) and TBAI (7 mg, 0.02 mmol) were added. The mixture was stirred at room temperature overnight. After the reaction completed (detected by TLC), diluted with 10 mL of water. The mixture was then extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with brine and dried over anhydrous Na₂SO₄, then the solution was concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain 23a (51 mg, 71%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.33 – 7.23 (m, 1H), 7.17 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 7.0 Hz, 1H), 5.91 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H), 5.33 (dq, J = 17.2, 1.5 Hz, 1H), 5.23 (dq, J = 10.4, 1.3 Hz, 1H), 4.63 (ddt, J = 5.8, 4.5, 1.4 Hz, 3H), 4.47 (dt, J = 8.0, 4.9 Hz, 2H), 3.91 – 3.73 (m, 2H), 2.39 (dt, J = 9.4, 7.4 Hz, 2H), 2.27 – 2.12 (m, 3H), 2.01 (dd, J = 23.1, 2.9 Hz, 13H), 1.80 – 1.51 (m, 10H), 1.44 (s, 9H), 1.04 – 0.80 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.69, 172.88, 172.31, 172.04, 171.14, 171.11, 131.82, 118.58, 80.74, 65.60, 62.24, 57.57, 52.59, 52.41, 48.56, 42.02, 40.48, 38.12, 36.48, 32.06, 30.77, 29.67, 28.20, 28.07, 27.40, 26.56, 26.11, 24.95, 23.20, 21.50, 19.07, 17.87. HRMS (ESI) calcd for C₃₉H₆₃N₄O₈ 715.4646 (M + H)⁺, found 715.4636.

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-3-methyl-1-oxo-1-(prop-2-yn-1-yloxy)butan-2-yl)amino)-5-oxopentanoate (23b).

To a solution of 22 (68 mg, 0.1 mmol) in 2 mL of dry DMF, NaHCO₃ (13 mg, 0.15 mmol), propargyl bromide (20 mg, 0.2 mmol) and TBAI (7 mg, 0.02 mmol) were added. The mixture solution was allowed to stir at room temperature overnight. After the reaction completed (detected by TLC), diluted with 10 mL of water. The mixture was then extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with brine and dried over anhydrous Na₂SO₄. The solution was concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1 to 20/1) to obtain 23b (49 mg, 69%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.29 – 7.17 (m, 2H), 6.76 (d, J = 7.0 Hz, 1H), 4.83 – 4.62 (m, 2H), 4.48 (dd, J = 8.5, 5.6 Hz, 2H), 4.32 (ddd, J = 10.8, 7.0, 4.4 Hz, 1H), 3.94 – 3.72 (m, 2H), 2.52 – 2.37 (m, 2H), 2.37 – 1.90 (m, 17H), 1.84 – 1.50 (m, 10H), 1.45 (s, 9H), 0.99 – 0.85 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.74, 172.91, 172.32, 172.04, 171.22, 170.72, 80.77, 77.36, 75.04, 62.28, 57.44, 52.60, 52.45, 52.28, 48.57, 42.04, 40.46, 38.13, 36.48, 32.09, 30.78, 29.68, 28.20, 28.07, 27.37, 26.57, 26.13, 24.97, 23.20, 21.50, 19.00. HRMS (ESI) calcd for C₃₉H₆₁N₄O₈ 713.4489 (M + H)⁺, found 713.4481.

(9H-Fluoren-9-yl)methyl (S)-2-(((S)-1-methoxy-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (25).

A solution of compound 24 (1.0 g, 3 mmol) and methyl L-leucinate (545 mg, 3 mmol) in CH₂Cl₂ (25 mL) was cooled to 0 °C with an ice bath. HOBt (460 mg, 3 mmol), HBTU (2.3 g, 6 mmol) and DIPEA (1 mL, 6 mmol) were added to the solution at the same temperature. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and the solution washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc= 5/1) to obtain compound 25 (1.2 g, 86%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.08 (d, J = 7.3 Hz, 1H), 4.72 – 4.20 (m, 5H), 3.66 (d, J = 47.5 Hz, 5H), 2.30 (d, J = 51.2 Hz, 1H), 1.97 (s, 2H), 1.75 – 1.52 (m, 4H), 0.90 (d, J = 5.9 Hz, 6H).

Methyl ((1R,3R)-adamantane-1-carbonyl)-L-prolyl-L-leucinate (26).

Compound 25 (1.2 g, 2.6 mmol) was added to a solution of HNEt₂ (1 mL) and MeCN (4 mL) at room temperature, and the solution was stirred at the same temperature for 1 h. When the reaction reached the end point (monitored by TLC), the solution was concentrated under reduced pressure to remove of the solvent. Then 10 mL of dry CH₂Cl₂ was added to dissolve the residue, and mixture solution was cooled to 0 °C with an ice bath. Compounds 1-adamantanecarboxylic acid (724, 2.6 mmol), HOBt (400 mg, 2.6 mmol), HBTU (2.0 g, 5.1 mmol) and DIPEA (1.3 mL, 8 mmol) were added to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 1/1) to obtain compound 26 (910 mg, 87%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.06 (s, 1H), 4.73 (dd, J = 7.9, 4.2 Hz, 1H), 4.53 (d, J = 7.1 Hz, 1H), 3.89 – 3.77 (m, 1H), 3.72 (t, J = 3.3 Hz, 4H), 2.34 – 2.19 (m, 1H), 2.15 – 1.84 (m, 12H), 1.67 (d, J = 34.8 Hz, 9H), 0.92 (q, J = 5.7 Hz, 6H).

((1R,3R)-Adamantane-1-carbonyl)-L-prolyl-L-leucine (27).

To a solution of 26 (910 mg, 2.2 mmol) in 10 mL of MeOH at 0 °C, LiOH∙H₂O (370 mg, 8.8 mmol) in 2 mL of H₂O was added. The solution was allowed to stir at room temperature overnight, and then acidified to pH = 3 by 1 N NaHSO₄. The mixture was extracted by CH₂Cl₂ and the organic layer was dried over Na₂SO₄. After concentration, compound 27 (720 mg, 84%) was obtained without further purification as a white foam. ¹H NMR (300 MHz, Chloroform-d) δ 7.41 (d, J = 8.1 Hz, 1H), 4.79 – 4.70 (m, 1H), 4.65 – 4.53 (m, 1H), 4.33 (s, 1H), 3.82 (dtt, J = 16.5, 10.2, 5.2 Hz, 2H), 2.27 – 1.85 (m, 13H), 1.80 – 1.54 (m, 9H), 0.94 (t, J = 6.2 Hz, 6H).

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-3-methyl-1-(((S)-oxiran-2-yl)methoxy)-1-oxobutan-2-yl)amino)-5-oxopentanoate (28a).

A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (680 mg, 2 mmol) and (S)-oxiran-2-ylmethanol (296 mg, 4 mmol) in CH₂Cl₂ (10 mL) was cooled to 0 °C with an ice bath. HOBt (612 mg, 4 mmol), EDCI (768 mg, 4 mmol) and DMAP (488 mg, 4 mmol) were added to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and the solution washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 10/1) to obtain compound ((S)-oxiran-2-yl)methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (650 mg, 82%). ¹H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.39 – 7.31 (m, 2H), 5.31 (d, J = 8.7 Hz, 1H), 4.59 – 4.34 (m, 4H), 4.26 (t, J = 7.0 Hz, 1H), 4.05 (dd, J = 12.2, 6.0 Hz, 1H), 3.24 (dq, J = 6.4, 3.1 Hz, 1H), 2.87 (t, J = 4.5 Hz, 1H), 2.69 (dd, J = 4.9, 2.6 Hz, 1H), 2.31 – 2.15 (m, 1H), 0.99 (dd, J = 16.9, 6.9 Hz, 6H).

Compound ((S)-oxiran-2-yl)methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (395 mg, 1 mmol) was added to a solution of HNEt₂ (1 mL) and MeCN (4 mL) at room temperature, and the reaction was stirred at the same temperature for 1 h. When the reaction reached the end point (monitored by TLC), the solution was concentrated under reduced pressure to remove the solvent. Then 10 mL of dry CH₂Cl₂ was added to dissolve the residue, and the solution was cooled to 0 °C with an ice bath. Fmoc-Glu(O^tBu)-OH (6) (420 mg, 1 mmol), HOBt (153 mg, 1 mmol), HBTU (758 mg, 2 mmol) and DIPEA (0.5 mL, 3 mmol) were added to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 2/1) to obtain compound 28a (460 mg, 79%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (td, J = 7.4, 1.2 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 5.80 (d, J = 7.7 Hz, 1H), 4.61 – 4.47 (m, 2H), 4.45 – 4.28 (m, 3H), 4.24 (t, J = 7.1 Hz, 1H), 4.02 (dd, J = 12.2, 6.0 Hz, 1H), 3.22 (s, 1H), 2.86 (t, J = 4.5 Hz, 1H), 2.68 (dd, J = 4.9, 2.6 Hz, 1H), 2.49 (q, J = 6.6 Hz, 2H), 2.26 (td, J = 7.0, 4.9 Hz, 1H), 2.16 – 1.93 (m, 2H), 1.49 (s, 9H), 0.98 (dd, J = 8.4, 6.9 Hz, 6H).

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (28b).

A solution of Fmoc-Glu(O^tBu)-OH (6) (420 mg, 1 mmol) and commercial available tert-butyl L-valinate (504 mg, 3 mmol) in CH₂Cl₂ (10 mL) was cooled to 0 °C with an ice bath. HOBt (153 mg, 1 mmol), HBTU (758 mg, 2 mmol) and DIPEA (0.5 mL, 2 mmol) were added to the solution at 0 °C. Then removed the ice bath, and the mixture solution was stirred at room temperature overnight. After the reaction was complete (detected by TLC), the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum, and the residue was purified by column chromatography (hexane/EtOAc = 5/1) to obtain compound 28b (550 mg, 95%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.37 – 7.30 (m, 2H), 6.88 (d, J = 8.6 Hz, 1H), 5.77 (d, J = 7.7 Hz, 1H), 4.49 – 4.36 (m, 3H), 4.26 (dt, J = 14.4, 7.0 Hz, 2H), 2.46 (q, J = 7.3 Hz, 2H), 2.26 – 1.95 (m, 3H), 1.48 (s, 18H), 0.95 (t, J = 6.9 Hz, 6H).

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(cyclohexyloxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (28c).

A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (680 mg, 2 mmol) and cyclohexanol (400 mg, 4 mmol) in 10 mL of CH₂Cl₂ was cooled to 0 °C with an ice bath. HOBt (612 mg, 4 mmol), EDCI (768 mg, 4 mmol) and DMAP (488 mg, 4 mmol) were added to the solution at 0 °C. Then the ice bath was removed, and the mixture solution was allowed to stir at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 10/1) to obtain compound cyclohexyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (500 mg, 57%). ¹H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (tt, J = 7.4, 1.2 Hz, 2H), 5.35 (d, J = 9.0 Hz, 1H), 4.87 (dt, J = 8.8, 4.6 Hz, 1H), 4.42 (d, J = 7.2 Hz, 2H), 4.36 – 4.22 (m, 2H), 2.20 (dt, J = 12.9, 6.7 Hz, 1H), 1.80 (d, J = 32.4 Hz, 4H), 1.41 (ddt, J = 31.5, 20.9, 12.0 Hz, 6H), 0.97 (dd, J = 18.5, 6.9 Hz, 6H).

Compound cyclohexyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (420 mg, 1 mmol) was added into a solution of HNEt₂ (1 mL) and MeCN (4 mL) at room temperature, and the solution was stirred at this temperature for 1 h. When the reaction reached the end point (monitored by TLC), the solution was concentrated under reduced pressure to remove the solvent. Then 10 mL of dry CH₂Cl₂ was added to dissolve the residue, and the solution was cooled to 0 °C with an ice bath. Fmoc-Glu(O^tBu)-OH (6) (420 mg, 1 mmol), HOBt (153 mg, 1 mmol), HBTU (758 mg, 2 mmol) and DIPEA (0.5 mL, 3 mmol) were added. Then removed the ice bath, and the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and the solution washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 5/1) to obtain compound 28c (510 mg, 84%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 6.97 (d, J = 8.6 Hz, 1H), 5.79 (d, J = 7.7 Hz, 1H), 4.93 – 4.75 (m, 1H), 4.51 (dd, J = 8.6, 4.6 Hz, 1H), 4.47 – 4.18 (m, 4H), 2.57 – 2.39 (m, 2H), 2.32 – 1.94 (m, 3H), 1.79 (d, J = 33.1 Hz, 4H), 1.49 (s, 15H), 0.96 (dd, J = 8.9, 6.8 Hz, 6H).

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-ethoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (28d).

Compound 28d (810 mg, 73%) was obtained by a similar synthetic process to prepare compound 28c as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (td, J = 7.4, 1.2 Hz, 2H), 7.03 – 6.91 (m, 1H), 5.77 (d, J = 7.7 Hz, 1H), 4.51 (dd, J = 8.6, 4.8 Hz, 1H), 4.43 – 4.37 (m, 2H), 4.36 – 4.16 (m, 4H), 2.56 – 2.38 (m, 2H), 2.29 – 2.08 (m, 2H), 1.99 (p, J = 7.2 Hz, 1H), 1.49 (s, 9H), 1.33 – 1.26 (m, 3H), 0.96 (dd, J = 8.9, 6.9 Hz, 6H).

tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-3-methyl-1-oxo-1-propoxybutan-2-yl)amino)-5-oxopentanoate (28e).

Compound 28e (870 mg, 77%) was produced by a similar synthetic process to prepare compound 28c as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.78 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 6.98 (d, J = 8.7 Hz, 1H), 5.77 (d, J = 7.7 Hz, 1H), 4.53 (dd, J = 8.6, 4.7 Hz, 1H), 4.44 – 4.19 (m, 4H), 4.13 (d, J = 11.9 Hz, 2H), 2.56 – 2.36 (m, 2H), 2.31 – 2.08 (m, 2H), 1.99 (dt, J = 14.3, 7.2 Hz, 1H), 1.76 – 1.65 (m, 3H), 1.49 (s, 9H), 0.96 (dd, J = 9.0, 6.9 Hz, 8H).

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-3-methyl-1-(((S)-oxiran-2-yl)methoxy)-1-oxobutan-2-yl)amino)-5-oxopentanoate (23c).

Compound 28a (300 mg, 0.5 mmol) was added to a solution of HNEt₂ (1 mL) and MeCN (4 mL) at room temperature, and the solution was stirred at this temperature for 1 h. When the reaction reached the end point (monitored by TLC), the solution was concentrated under reduced pressure to remove of the solvent. Then 10 mL of dry CH₂Cl₂ was added to dissolve the residue, and the solution was cooled to 0 °C with an ice bath. Then, compound 27 (204 mg, 0.5 mmol), HOBt (153 mg, 1 mmol), HBTU (379 mg, 1 mmol) and DIPEA (0.2 mL, 3 mmol) were added. Then removed the ice bath, and the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (hexane/EtOAc = 5/1) to obtain compound 23c (290 mg, 79%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.30 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 6.71 (d, J = 6.9 Hz, 1H), 4.65 – 4.56 (m, 1H), 4.55 – 4.35 (m, 3H), 4.28 (ddd, J = 10.6, 6.8, 4.4 Hz, 1H), 4.02 (dd, J = 12.2, 5.8 Hz, 1H), 3.81 (ddt, J = 20.3, 7.5, 3.8 Hz, 2H), 3.31 – 3.14 (m, 1H), 2.81 (t, J = 4.5 Hz, 1H), 2.66 (dd, J = 4.9, 2.6 Hz, 1H), 2.37 (dd, J = 9.5, 7.2 Hz, 2H), 2.25 – 1.83 (m, 17H), 1.72 (d, J = 5.2 Hz, 8H), 1.43 (s, 9H), 1.02 – 0.83 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.77, 172.82, 172.40, 172.03, 171.27, 171.12, 80.71, 77.26, 65.05, 62.41, 57.62, 52.73, 52.59, 49.14, 48.61, 44.73, 42.03, 40.40, 38.12, 36.56, 36.46, 32.11, 30.62, 28.18, 28.05, 27.27, 26.65, 25.01, 23.18, 21.48, 19.04, 17.98. HRMS (ESI) calcd for C₃₉H₆₃N₄O₉ 731.4590 (M + H)⁺, found 731.4586.

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (23d).

Compound 23d (116 mg, 72%) was produced by a similar synthetic process to prepare compound 23c from 28b and 27 as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.20 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 7.1 Hz, 1H), 4.69 – 4.57 (m, 1H), 4.46 (td, J = 8.0, 5.3 Hz, 1H), 4.31 (td, J = 8.0, 7.1, 4.7 Hz, 2H), 3.92 – 3.67 (m, 2H), 2.48 – 2.28 (m, 2H), 2.23 – 1.91 (m, 16H), 1.80 – 1.52 (m, 9H), 1.44 (d, J = 5.6 Hz, 18H), 0.97 – 0.84 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.58, 172.80, 172.22, 171.95, 170.88, 170.42, 81.53, 80.66, 62.13, 58.00, 52.54, 52.33, 48.50, 42.02, 40.56, 38.13, 36.49, 32.03, 30.90, 28.21, 28.06, 28.00, 27.52, 24.90, 23.17, 21.53, 18.97, 17.78. HRMS (ESI) calcd for C₄₀H₆₇N₄O₈ 731.4953 (M + H)⁺, found 731.4945.

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-(cyclohexyloxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (23e).

Compound 23e (310 mg, 80%) was obtained by a similar synthetic process to prepare compound 23c from 28c and 27 as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.21 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 7.1 Hz, 1H), 4.83 (tt, J = 8.9, 3.8 Hz, 1H), 4.71 – 4.61 (m, 1H), 4.47 (ddd, J = 11.4, 8.3, 5.4 Hz, 2H), 4.33 (ddd, J = 9.7, 7.0, 4.3 Hz, 1H), 3.93 – 3.71 (m, 2H), 2.41 (dtd, J = 16.4, 9.2, 7.2 Hz, 2H), 2.26 – 1.92 (m, 16H), 1.90 – 1.58 (m, 13H), 1.46 (s, 15H), 1.02 – 0.86 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.65, 172.91, 172.22, 171.94, 170.98, 170.77, 166.60, 80.74, 73.47, 62.15, 57.60, 52.55, 52.37, 48.52, 42.04, 40.53, 38.14, 36.50, 32.08, 31.51, 30.90, 28.22, 28.08, 27.58, 25.32, 24.93, 23.64, 23.21, 21.50, 19.01, 17.78. HRMS (ESI) calcd for C₄₂H₆₉N₄O₈ 757.5110 (M + H)⁺, found 757.5106.

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-1-ethoxy-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (23f).

Compound 23f (260 mg, 74%) was obtained by a similar synthetic process to prepare compound 23c from 28d and 27 as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.24 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 7.1 Hz, 1H), 4.64 – 4.56 (m, 1H), 4.51 – 4.37 (m, 2H), 4.29 (ddd, J = 9.6, 7.0, 4.5 Hz, 1H), 4.15 (qt, J = 7.1, 3.6 Hz, 2H), 3.89 – 3.68 (m, 2H), 2.35 (q, J = 7.5 Hz, 2H), 2.15 (dddd, J = 14.0, 12.1, 7.5, 4.2 Hz, 3H), 2.05 – 1.91 (m, 12H), 1.79 – 1.46 (m, 9H), 1.41 (s, 9H), 1.24 (t, J = 7.1 Hz, 4H), 0.94 – 0.83 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 177.60, 172.76, 172.30, 172.02, 171.35, 171.07, 80.65, 62.23, 60.94, 57.55, 57.47, 52.58, 52.38, 48.53, 42.00, 40.49, 38.12, 36.46, 31.99, 31.73, 30.74, 29.62, 28.19, 28.03, 28.01, 27.41, 26.62, 26.04, 24.92, 23.13, 21.52, 18.98, 17.87, 17.62, 14.15. HRMS (ESI) calcd for C₃₈H₆₃N₄O₈ 703.4640 (M + H)⁺, found 703.4636.

tert-Butyl (4S)-4-((2S)-2-((2S)-1-((1R,3R)-adamantane-1-carbonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-5-(((S)-3-methyl-1-oxo-1-propoxybutan-2-yl)amino)-5-oxopentanoate (23g).

Compound 23g (274 mg, 77%) was obtained by a similar synthetic process to prepare compound 23c from 28e and 27 as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.36 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 6.66 (d, J = 6.6 Hz, 1H), 4.54 – 4.34 (m, 3H), 4.27 – 4.15 (m, 1H), 4.13 – 3.97 (m, 2H), 3.83 (p, J = 5.7, 4.5 Hz, 2H), 2.34 (tt, J = 6.9, 2.7 Hz, 2H), 2.26 – 1.81 (m, 16H), 1.79 – 1.47 (m, 11H), 1.40 (s, 9H), 0.99 – 0.81 (m, 15H). ¹³C NMR (75 MHz, CDCl₃) δ 177.97, 173.09, 172.69, 172.47, 172.02, 171.44, 170.38, 80.73, 66.83, 62.88, 57.74, 52.96, 52.88, 48.71, 41.99, 40.12, 38.04, 36.41, 31.97, 30.66, 28.14, 28.01, 27.34, 26.97, 26.11, 24.98, 23.04, 21.82, 21.53, 18.98, 17.97, 10.32. HRMS (ESI) calcd for C₃₉H₆₅N₄O₈ 717.4797 (M + H)⁺, found 717.4794.

1-(tert-Butyl) 5-methyl ((benzyloxy)carbonyl)-L-prolyl-L-leucyl-L-glutamate (30).

To a solution of compound 10 (326 mg, 1 mmol) and H-Glu(OMe)-O^tBu•HCl (29) (254 mg, 1 mmol) in CH₂Cl₂ (10 mL), HOBt (135 mg, 1 mmol), HBTU (758 mg, 2 mmol) and DIPEA (1.3 mL, 8 mmol) were added to the solution at 0 °C. Then the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain 30 (540.8 mg, 90%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.37 (d, J = 4.3 Hz, 5H), 7.01 – 6.21 (m, 2H), 5.17 (s, 2H), 4.57 – 4.27 (m, 3H), 3.67 (s, 3H), 3.54 (s, 2H), 2.50 – 1.88 (m, 8H), 1.47 (s, 12H), 0.91 (t, J = 6.5 Hz, 6H). ¹³C NMR (75 MHz, Chloroform-d) δ 173.27, 171.63, 170.42, 128.52, 128.12, 127.85, 82.26, 67.39, 60.66, 52.23, 51.93, 51.71, 47.11, 40.55, 29.98, 27.95, 27.42, 24.88, 22.91, 21.84. . HRMS (ESI) calcd for C₂₉H₄₃N₃O₈ 561.3050 (M + H)⁺, found 561.3042.

Benzyl (S)-2-(((S)-1-(((S)-1-amino-3-methyl-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (31).

To a solution of compound 10 and compound 7 (42 mg, 0.27 mmol) in 5 mL of CH₂Cl₂, HOBt (36 mg, 0.27 mmol), HBTU (204 mg, 0.54 mmol) and DIPEA (0.18 mL, 1.08 mmol) were added to the solution at 0 °C. Then, the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain 31 (112 mg, 90%) as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 11.0 Hz, 1H), 7.33 (d, J = 18.9 Hz, 7H), 7.06 (s, 1H), 5.27 – 4.69 (m, 2H), 4.31 – 4.00 (m, 3H), 3.39 (d, J = 20.2 Hz, 2H), 2.13 (s, 1H), 1.89 – 1.35 (m, 6H), 1.17 (t, J = 7.1 Hz, 1H), 1.09 – 0.55 (m, 12H). ¹³C NMR (75 MHz, DMSO-d₆) δ 173.09, 172.48, 172.10, 172.06, 137.45, 128.82, 128.64, 128.21, 127.91, 127.31, 66.32, 66.16, 60.20, 59.44, 57.59, 57.41, 51.90, 51.73, 47.58, 40.75, 31.59, 31.30, 31.11, 30.43, 24.63, 24.29, 23.42, 21.99, 21.83, 19.68, 18.17. HRMS (ESI) calcd for C₂₄H₃₆N₄O₅ 460.2686 (M + H)⁺, found 460.2682.

Benzyl (S)-2-(((S)-1-(((S)-1-methoxy-3-methyl-1-oxobutan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (32).

A solution of compound 10 (100 mg, 0.27 mmol) and 14 (42 mg, 0.27 mmol) in CH₂Cl₂ (5 mL) was cooled to 0 °C with an ice bath, followed by adding HOBt (36 mg, 0.27 mmol), HBTU (204 mg, 0.54 mmol) and DIPEA (0.18 mL, 1.08 mmol) to the solution at 0 °C. Then removed the ice bath, and the mixture solution was stirred at room temperature overnight. After the reaction finished (monitored by TLC), additional 25 mL of CH₂Cl₂ was added to the reaction solution and then washed with 1 N NaHSO₄, saturated NaHCO₃ and brine. The organic solution was dried over anhydrous Na₂SO₄, and then concentrated under vacuum and the residue was purified by column chromatography (CH₂Cl₂/MeOH = 50/1) to obtain 32 (108 mg, 84%) as a white solid. ¹H NMR (300 MHz, Chloroform-d) δ 7.32 (d, J = 22.7 Hz, 5H), 6.68 (dd, J = 107.1, 65.7 Hz, 2H), 5.18 (s, 2H), 4.64 – 4.18 (m, 3H), 3.74 (s, 3H), 3.51 (d, J = 16.3 Hz, 2H), 2.40 – 1.53 (m, 8H), 1.12 – 0.67 (m, 12H). ¹³C NMR (75 MHz, Chloroform-d) δ 172.06, 171.60, 128.53, 128.13, 127.85, 76.98, 67.40, 60.60, 57.17, 52.06, 47.09, 40.24, 31.10, 28.43, 24.84, 24.61, 22.84, 21.94, 18.90, 17.72. HRMS (ESI) calcd for C₂₅H₃₇N₃O₆ 475.2682 (M + H)⁺, found 475.2679.

Molecular Docking Method.

The molecular docking study was carried out using Schrödinger Small-Molecule Drug Discovery Suite (Schrödinger, LLC, New York, NY, 2020). FGF14:Na_v1.6 homology model was generated using the FGF13:Na_v1.5: CaM ternary complex crystal structure (PDB code: 4DCK) as a template. The FGF14:Na_v1.6 homology model was prepared with Schrödinger Protein Preparation Wizard using default settings. During this step, hydrogens were added, crystal waters were removed, and partial charges were assigned using the OPLS-2005 force field. The SiteMap calculation was investigated and a potential binding site was identified on the PPI of FGF14:Na_v1.6. The chain of Na_v1.6 was excluded and the grid center was chosen on the center of this previous identified binding site with a grid box in size of 24 Å on each side. The 3D structure of ligand 21a was created using Schrödinger Maestro and a low energy conformation was calculated using LigPrep. Docking was employed with Glide using the SP protocol. Docked poses were incorporated into Schrödinger Maestro for a ligand-receptor interactions visualization. The top docked pose of 21a was selected and superimposed with FGF14:Na_v1.6 complex homology model for an overlay analysis.

DNA Constructs.

The CLuc-FGF14-1b, CD4-Na_v1.6-NLuc, pET28a-FGF14, and pET30a-Na_v1.6 constructs were engineered and characterized as previously described.^{36, 47, 50, 52–54} The LCA constructs encoded for the expression of recombinant human FGF14-1b (aa 1–252) and recombinant human Na_v1.6 C-terminal tail (aa 1763–1976) protein. The plasmid pGL3 expressing full length Firefly (Photinus pyralis) luciferase was a gift from Dr. P. Sarkar (Department of Neurology, UTMB). cDNAs encoding FGF14 (accession number NP_787125; aa 64–252) or the C-terminal tail of Na_v1.6 (accession number #NP_001171455; aa 1767–1912) were sub-cloned into suitable pET bacterial expression vectors (pET28a-FGF14; pET30a-Na_v1.6) with a 6X His-tag at the N-terminal site; these plasmids were a gift of Dr. Moosa Mohammadi (NYU, Langone Medical Center).

Cell Culture.

HEK293 cells were maintained in DMEM and F-12 (Invitrogen), supplemented with 0.05% glucose, 0.5 mM pyruvate, 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), and incubated at 37 °C with 5% CO₂. The double stable HEK293 cell line expressing CD4-Na_v1.6-C-tail-NLuc and CLuc-FGF14 was previously described^{47, 48} and was maintained using selective antibiotics (0.5 mg/mL G418 and 5 μg/mL puromycin). HEK293 cells stably expressing the human Na_v1.6 channel (hereafter referred to as HEK-Na_v1.6 cells) were maintained similarly except for the addition of 500 μg/mL G418 (Invitrogen) to maintain stable Na_v1.6 expression. Cells were transfected at 80–90% confluence with equal amount (1 μg each) of plasmid pairs using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. HEK-Na_v1.6 cells were washed and re-plated at very low density prior to electrophysiological recordings.^{36, 47, 50, 55}

Split-Luciferase Complementation Assay (LCA).

Cells were trypsinized (using a 0.04% Trypsin:EDTA mixture dissolved in PBS), centrifuged, triturated in fresh medium, and seeded in white, clear-bottom 96-well tissue culture plates (Greiner Bio-One) using 200 μL of medium per well. For transfected cells, the trypsinization occurred 24 h post-transfection. The cells were incubated for 24 h and then the growth medium was replaced with 100 μL of serum-free, phenol red–free DMEM/F12 medium (Invitrogen) containing either 0.5% DMSO alone (vehicle; n = 32 replicates per plate) or compounds (50 μM for screening and 0.25–100 μM for dose responses; n =4 replicates per treatment condition per plate) dissolved to a final concentration of 0.5% DMSO, with a minimum of 3 independent experiments per compound. The bioluminescence reaction was initiated by dispensing 100 μL of D-luciferin substrate (1.5 mg/mL dissolved in PBS) using a Synergy^™ H1 Multi-Mode Microplate Reader (BioTek, Agilent). Luminescence readings were performed at 2 min intervals for 20 min with an integration time of 0.5 s. Cells were maintained at 37 °C throughout the measurements. Signal intensity for each well was calculated as a mean value of peak luminescence; the calculated values were expressed as percentage of mean signal intensity in the control samples from the same experimental plate. Detailed methods for LCA can be found in previous studies.^{36, 44, 47, 50, 53, 56} To determine the most effective compounds, those with scores above or below two standard deviations (relative to per plate DMSO controls) were defined as hits. Hits were prioritized and further pursued based on the magnitude of effect.

Dose-response curves were obtained using GraphPad Prism 8 by fitting the data with a non-linear regression:

$$A+\frac{B-A}{1+{10}^{\text{log}\left(x_0-x\right)}H}$$ (6)

where x is log10 of the compound concentration in M, x₀ is the inflection point (EC₅₀ or IC₅₀), A is the bottom plateau effect, B is the top plateau effect, and H is the Hill slope.

Cell Viability Assay.

The CellTiter-Blue® Cell Viability (CTB) assay (Promega) was used as a counter screen to rule out compound toxicity, as described previously.⁴⁸ Immediately following LCA luminescence reading from treated cells, 30 μL of 1X CTB reagent was dispensed into 96-well plates; plates were then incubated overnight (16 h) at 37 °C, and fluorescence was detected using the Synergy H1 reader (excitation λ = 560 nm, emission λ = 590 nm). Cell viability was expressed as percent of the mean fluorescent signal intensity of on-plate negative controls.

Protein Expression and Purification.

The two plasmids for protein expression and purification of FGF14 and Na_v1.6 C-tail have been previously described^{50, 55} and were transformed into E. coli BL21 (DE3) pLys (Invitrogen). Cells were grown until OD₆₀₀ = 0.7, and the recombinant proteins were expressed after induction with 0.1 mM isopropyl thio-β-D-galacto-pyranoside (IPTG) for 24 h at 16 °C. Cells were harvested and lysed by sonication at 4 °C in lysis/binding buffer containing following components (mM): 10 sodium phosphate (prepared from 0.5 M of Na₂HPO₄ and NaH₂PO₄), 25 HEPES, 150 NaCl, phenyl methyl sulphonyl fluoride (PMSF) 0.1, CHAPS 0.1% pH 7.0 (for FGF14), and with glycerol 10% (for Na_v1.6 C-terminal tail) pH 7.5. The respective proteins were centrifuged at 40,000 × g for 1 h at 4 °C. For purification of FGF14, the supernatant was applied to pre-equilibrated heparin and the proteins were then eluted with NaCl 0.2–2.0 M (sodium phosphate 10 mM, NaCl 0.2–2.0 M, pH 7.0) buffer. For purification of Na_v1.6 C-terminal tail, the supernatant was first applied to a cobalt column (Thermo Fisher Scientific) and eluted with imidazole (200 mM). The Na_v1.6 C-terminal tail was further purified using HiTrap QFF-sepharose column (GE Healthcare) using a buffer containing Tris-HCl 50 mM and eluted with NaCl (10–500 mM) at pH 7.5. Finally, all concentrated proteins were purified on an AKTA FPLC using a Superdex 200 HiLoad 16 × 60 column and equilibrated in Tris-HCl 50 mM + NaCl 150 mM, pH 7.5 (GE Healthcare). Protein concentrations were determined using UV absorbance with a Thermo NANODROP.

Protein Thermal Shift Assay.

The protein thermal shift (PTS) assay was used to identify compounds that interacted with purified FGF14 and Na_v1.6 C-terminal tail protein. Melting (denaturation) of globular proteins exposes hydrophobic regions that interact with the fluorescent dye, resulting in increased fluorescence as detected by the PCR detection system.⁴⁹ Changes in protein thermal stability by ligand binding can be detected by shifts in the temperature at which fluorescent peaks are observed compared to protein in the absence of ligand, enabling the estimation of change in melting temperature (ΔT_M). Reactions were prepared in 96-well PCR plates using the PTS Dye Kit (Applied Biosystems, Life Technologies) as per manufacturer instructions, and the assay was conducted on a QuantStudio 3 rtPCR System. Each well included a total reaction volume of 20 μL comprised of 2 μg of either FGF14 or Na_v1.6 C-terminal tail protein, 1X dye, and peptidomimetics (50 μM; n = 4 replicates per compound) or 0.5% DMSO alone (control; n = 8 replicates per protein per plate) in PBS, and compounds were assessed over a minimum of 2 independent experiments. Additional per plate controls (n = 4 replicates per condition) included buffer and dye alone (no protein control) and compounds alone (in the absence of protein) to assess potential interactions between peptidomimetics and fluorescent dye. Plates with an unacceptably high degree of variability (> ±2 °C) or with protein-only controls that significantly deviated from consistently observed T_M averages (i.e., 49 °C for Nav1.6 C-terminal tail and 60 °C for FGF14) were excluded from analysis, and the conditions retested. Plates were heated from 25 to 99 °C at a ramp rate of 0.05 °C/s with ROX as the selected reporter. The Boltzmann method was used to obtain protein T_M, and the change in melting temperature was calculated using the mean of per plate controls for each respective protein (FGF14 or Na_v1.6 C-terminal tail).

Surface Plasmon Resonance Spectroscopy.

SPR experiments were performed on a Biacore T100 instrument (GE Healthcare). Proteins were immobilized on CM5 sensor chips using 10 mM sodium acetate buffer (pH 5.5) with the Amine Coupling Kit (GE Healthcare) as per the manufacturer’s instructions, to a final RU value of 17,896 for FGF14 and 6,800 for Na_v1.6 C-terminal tail. No protein was coupled to the control flow channels of the chip (Lanes 1 and 3). The interaction of experimental compounds against FGF14 and Na_v1.6 proteins were studied at 25 °C using a flow rate of 50 μL/min. Compounds were serially diluted (0.195–200 μM) in PBS supplemented with Tween-20 0.005% and 2% DMSO. Each sample was injected over the chip for 120 s followed by a dissociation period of 150 s and finally chip surface regeneration (600 mM NaCl, 5% DMSO) for 120 s. Each compound was tested over at least two independent experiments with concentrations of 0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 μM, including 12.5 μM, 25 μM, and blanks (buffer alone) in duplicate. A DMSO calibration was performed for each experiment 1 [1.5–2.8% (v/v) DMSO] to correct for bulk refractive index shifts.⁵⁷ Protein:protein and protein:protein-ligand complex studies were conducted similarly, with the exception that increasing concentrations (15.6, 31.3, 62.5, 125, 250, 500, 750, 1000, 1250, and 1500 nM) of FGF14 in complex with either DMSO alone (2.1%) or compounds (10 μM) were flown over a CM5 sensor chip with Na_v1.6 C-terminal tail bound (934 RU); all conditions were maintained with a final concentration of 2.1% DMSO. For each compound injection, nonspecific responses (“0 μM” solution prepared similarly to experimental compound samples) for each corresponding experiment were subtracted from compound sensorgrams/traces prior to data analysis. Kinetic data were analyzed using the Biacore T100 Analysis software. Following visual inspection of the binding curves, the equilibrium constant (K_D) was calculated using two methods: (1) maximal responses were plotted against compound concentration, and the steady state K_D was calculated from the fitted saturation binding curve; (2) a kinetic analysis of each ligand/analyte interaction was obtained by fitting the response data to the simplest Langmuir 1:1 interaction model (K_D = k_off/k_on). The kinetic constants generated from the fitted binding curves were assessed for accuracy based on the distribution of the residuals (even and near zero to baseline). Compounds with incalculable k_off or k_on values (i.e., due to either lack of compound binding, or k_on and/or k_off approaching limits of detection) toward the binding partner are denoted as non-determinable (ND). Compounds failing to achieve saturation of binding over the concentration range tested are reported as > 200 μM. Graphs were plotted in GraphPad Prism 8 Software (La Jolla, CA).

Electrophysiology.

HEK-Na_v1.1, HEK-Na_v1.2, and HEK-Na_v1.6 cells adhered to glass cover slips were transferred to a recording chamber containing an extracellular recording solution comprised of the following salts (mM): 140 NaCl, 3 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, 10 glucose (Sigma-Aldrich, St. Louis, MO, USA; pH = 7.3), and either 0.1% DMSO or experimental. After at least 30 min incubation, recordings were performed with borosilicate glass pipettes (World Precision Instruments, Sarasota, FL, USA; resistance = 3–6 MΩ) that were fabricated using a two-step vertical puller PC-100 (Narishige, Amityville, NY, USA) and filled with an internal solution comprised of the following salts (mM): 130 CH₃O₃SCs, 1 EGTA, 10 NaCl, and 10 HEPEs (Sigma-Aldrich; pH = 7.3). After giga-seal formation and cell membrane rupture, membrane capacitance and series resistance were estimated and compensated for by 70–80% using dial setting on the amplifier. After compensation, recordings were performed using an Axopatch 200b amplifier (Molecular Devices, Sunnyvale, CA, USA). Detailed information regarding electrophysiological protocols has been reported.³⁵ Data acquisition and stimulation were performed with a DigiData 1322A Series interface and pClamp 9 software (Molecular Devices). Data were filtered at 5 kHz and digitized at 20 kHz. Acquired electrophysiological data was then analyzed as previously described using pClamp 10 (Molecular Devices)³⁵ and GraphPad Prism 8 software (La Jolla, CA, USA). Results were expressed as the mean ± the standard error of the mean (SEM). A Student’s t-test was used to determine statistical significance, with p < 0.05 being considered statistically significant.

Table 7.

Characterization of FGF14:Na_v1.6 binding in the presence of 21a by SPR.

	KD (nM)*	kon (M−1 s−1)	koff (s−1)
FGF14:Nav1.6	228.50 ± 57.54	2.18×104 ± 1.6×102	1.71×10−3 ± 1.02×10−5
FGF14+21a:Nav1.6	1418.65 ± 134.7	5.88×104 ± 2.9×102	3.68×10−3 ± 6.70×10−5

^*The equilibrium dissociation constants (K_D) were calculated using data shown in Figure 7. Each K_D is an average of that calculated using the simplest Langmuir 1:1 interaction model (K_D = k_off/k_on) and the steady-state saturation model. ND, non-determinable.

ABBREVIATIONS USED

Na_v

Voltage-gated sodium

CNS

central nervous system

PNS

peripheral nervous system

SE

status epilepticus

mEC

medial entorhinal cortex

PPI

protein-protein interaction

FGF14

fibroblast growth factor 14

MSNs

medium spiny neurons

PTS

protein thermal shift

SPR

surface plasmon resonance

LCA

split-luciferase complementation assay

RLVs

relative luminescence values

FLL

full-length luciferase

CTB

CellTiter-Blue® Cell Viability

SAR

structure-activity relationship

Cbz

1-Hydroxybenzotriazole

HOBt

benzyloxycarbonyl

HBTU

2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

DIPEA

N,N-Diisopropylethylamine

DMF

N,N-dimethylformamide

DMAP

4-dimethylaminopyridine

EDCI

1-ethyl-(3-dimethylaminopropyl)carbonyldiimide