Small Peptides Targeting BACE-1, AChE, and A-β Reversing Scopolamine-Induced Memory Impairment: A Multitarget Approach against Alzheimer’s Disease

Abstract

Based on the biochemical understanding of Alzheimer’s disease, here, we report the design, synthesis, and biological screening of a series of compounds against this neuro-disorder. Adopting the multitarget approach, the catalytic processes of BACE-1 and AChE were targeted, and thereby, compounds 15, 22, 25, 26, 27, and 30 were identified with IC₅₀ in the submicromolar range against these two enzymes. Further, compounds 15 and 25 displayed more than 50% inhibition of β-amyloid aggregation. Implying their physiological use, the compounds exhibited appreciable biological membrane permeability as observed through the parallel artificial membrane permeability experiment. Supporting these results, treatment of the mice with the test compounds reversed their scopolamine-affected memory impairment, where the highest healing effect was seen in the case of compound 25. Overall, the combination of molecular modeling and experimental studies provided highly effective molecules against Alzheimer’s disease.

1. Introduction

Neuro-degenerative disorders including Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis severely hamper the functioning of the CNS.¹ Psychiatric conditions including schizophrenia and clinical depression are also associated with brain dysfunctions.² Moreover, the incidence of cognitive impairment leads to behavioral changes, and eventually, under chronic conditions, it may develop into Alzheimer’s disease (AD), which can prove fatal.³ With ∼25 million people suffering from AD worldwide, the number is expected to double by 2050.⁴ Since the disease is more prevalent in the aged population, increased life expectancy is also adding to the number of AD cases.⁵ Despite the severe nature of this neurological disorder, there is no cure for the disease to date, while the current treatments for Alzheimer’s disease merely address its symptoms. Even the reasons for the disease’s origin are still not clearly known though complex molecular pathways including neurotransmitters,⁶ oxidative stress,⁷ nitrosative stress,⁷ eicosanoids,⁸ and inflammatory cytokine mediators⁹ are expected to play key roles in the emergence of Alzheimer’s disease.

While focusing on these biochemical pathways that contribute to the initiation and propagation of the disease, various treatment strategies targeting BACE-1,¹⁰⁻¹⁵ AChE,¹⁶⁻¹⁸ and kinases¹⁹⁻²¹ are adopted to address AD. However, due to the multifactorial nature of this neuro-disorder, several single-target guided AD drugs did not pass through the clinical trials. It was, therefore, planned to develop new chemical entities that can target multiple AD-associated enzymes. We present here in vitro and in vivo experiments with a set of molecules against the AD-related cellular targets and identify some highly potent molecules.

2. Results and Discussion

2.1. Selection of Targets and the Design Strategy for New Molecules

The most widely accepted pathologic reason for the appearance of AD symptoms is the accumulation of certain mass in the brain, which was later identified as amyloid β protein (Aβ)_1–40/42. This protein is formed by the action of BACE-1 on APP generating the soluble amyloid protein β (sAPPβ) and the subsequent action of γ-secretase on sAPPβ (red part, Figure 1).^22,23 It is observed that out of the two isoforms of β-secretase—the β-site APP cleaving enzyme 1 (BACE-1) and the β-site APP cleaving enzyme 2 (BACE-2)—present in the brain, BACE-1 plays a predominant role in the commencement of AD.²⁴ Mechanistically, the cleavage of APP at the Asp–Met juncture is accomplished by the participation of Asp32 and Asp228 (catalytic diad) of the active site of the enzyme (Figure 1A). The activation of an enzyme-bound water molecule by one of the two catalytic aspartates enables it to react at the carbonyl carbon of the amide group, thus releasing the products of peptide cleavage.²⁵ It is, therefore, inferred that the BACE-1 inhibitors must target the catalytic diad of the enzyme.

Figure 1.

(A) Mechanism of APP breakdown (catalytic diad) under the effect of α-secretase (gray-blue part) and β-secretase (gray-red part). The action of β-secretase followed by γ-secretase is responsible for the generation of the amyloid β protein (Aβ)_1–40/42—the causative agent of AD. (B) Mechanism of AChE-mediated hydrolysis of ACh showing a catalytic triad.

Another factor supplementing BACE-1 in the initiation/progression of AD is the reduced level of neurotransmitters, acetylcholine (ACh), in the brain. Since the ACh level is reduced due to the cholinesterase (AChE) activity, this enzyme served as the potential target of anti-AD drugs.²⁶ AChE-based hydrolysis of ACh is assisted by the Glu327, His440, and Ser200 triad (Figure 1B).²⁷ Apart from the active site, AChE contains a peripheral anionic site (PAS), which binds different kinds of ligands/uncompetitive inhibitors, thus changing the conformation of the active site and inhibiting the activity of AChE.²⁸ Hence, developing AChE inhibitors capable of interacting with its Glu327, His440, and Ser200 triad is one of the strategic approaches for the treatment of AD.

Therefore, keeping in view the two targets of AD-treatment agents, herein, the focus was on the design of dual inhibitors of BACE-1 and AChE. As per literature reports, structurally diverse BACE-1 and AChE inhibitors (Figure 2A)²⁹⁻³⁴ consist of fused bicyclic systems such as indene, chromone, acridine, and indole, while pyrrolidine and piperidine also comprise an integral part of their structure. Since the active sites “diad” and “triad” of BACE-1 and AChE are constituted by polar residues, it was envisaged that the inhibitors of these enzymes must have an optimum log P and total polar surface area (TPSA) so that they can easily cross the BBB and interact in the active site pockets of the enzymes. Owing to the presence of polar groups in peptides and the possibility of their derivatization through N-/C- termini, it was planned to develop small peptides for targeting BACE-1 and AChE. Moreover, for reducing the molecular flexibility and imparting a bent structure (controlled surface area), proline was chosen as the template, whereas hydrophobic moieties were tagged at the N-/C-termini to adjust the physicochemical properties of the molecules (Figure 2B).

Figure 2.

(A) Multitarget inhibitors of AD enzymes and (B) design of new molecules.

Design of the molecules was validated by their molecular docking in the active site pockets of BACE-1 and AChE (Figures S1–S45 and Table S1). A number of H-bond and hydrophobic interactions between the molecules and the two enzymes were observed. Compound 27 interacts through the H-bond with G230 and D228 of BACE-1. It also makes two H-bonds with the water molecules in the active site (Figure 3A). Similarly, H-bonding of 25 with T76, R128, and water molecules was observed (Figure 3B). Appreciable interactions of the newly designed compounds were also observed in the active site pocket of AChE (Figure 3C,D). Both the compounds 15 and 25 were found to interact with H447 of the catalytic triad of AChE. Compound 25 orients in the active site of BACE-1 in a way that it interacts with T84 through its one arm and forms a H-bond with water molecules from its other end.

Figure 3.

Binding mode of (A) Cbz-Gly-Pro-Ala-O-crotyl (27) in BACE-1 (PDB ID 6EQM),³⁵ (B) Cbz-Gly-Pro-Ala-O-cinnamyl (25) in BACE-1, (C) Iso-Gly-Pro-Ala-O-Allyl (15) in AChE, and (D) 25 in AChE (PDB ID 4EY6)³⁶ (also see the Supporting Information).

2.2. Chemistry

N-Boc protected l-proline 9 was coupled with l-Ala-OMe in the presence of triethylamine (TEA) and ethyl chloroformate (ECF) in tetrahydrofuran (THF) to procure compound 10. The Boc group of 10 was removed with trifluoroacetic acid (TFA) to obtain compound 11. Compound 12, synthesized by the reaction of iso-butyric acid and Gly-OH, was coupled with 11 to get compound 13. Compound 14, obtained by the ester group hydrolysis of 13, was re-esterified with allyl bromide/crotyl bromide/dimethyl bromide/propargyl bromide/geranyl bromide/cinnamyl bromide, and thereby compounds 15–19 were obtained (Scheme 1).

Scheme 1. Syntheis of Compounds 15–27.

Reaction conditions: (i) dioxane/H₂O (2:1), NaOH, rt,1.5 h; (ii) TEA, ECF, THF, −10 °C to rt, 8 h; (iii) TFA, DCM, 8 h; (iv) acetone/H₂O (3:2), NaOH, rt; (v) ACN, K₂CO₃, 60 °C, 12 h; (vi) CbzCl, NaOH, H₂O.

Further, Gly-OH was reacted with CbzCl, and the resulting compound 20 was coupled with 11 to get compound 21. Ester hydrolysis of 21 gave compound 22, which was derivatized with allyl/propargyl/crotyl/cinnamyl/geranyl/dimethylallyl bromides to obtain the series of compounds 23–27. These esterifying agents and the isobut/Cbz groups helped in balancing the TPSA and logP of the compounds. For the synthesis of compound 30, boc-Gly 28 was coupled with dipeptide 11 to get compound 29, which was deprotected of the boc group to obtain 30 (Scheme 2). For comparison, due to providing more rigidity and hydrophobicity (less TPSA) to the molecule, compound 33 was included in the present studies. Synthesis of 33 was accomplished by the reaction of (S)-binol with propargyl bromide and the coupling of the resulting compound 31 with cinnamyl azide (Scheme 3).

Scheme 2. Synthesis of Compounds 29 and 30.

Reaction conditions: (i) dioxane/H₂O (2:1), NaOH, rt,1.5 h; (ii) TEA, ECF, THF, −10 °C to rt, 8 h; (iii) TFA, DCM, 8 h.

Scheme 3. Synthesis of Compound 33.

Reaction conditions: (i) K₂CO₃, DMF, 60 °C; (ii) EtOH/H₂O (9:1), CuSO₄·5H₂O, sodium ascorbate.

2.3. Biological Studies

2.3.1. BACE-1 Inhibitory Activities

BACE-1 inhibitory activities of the compounds were determined by using bioassay kits. Among the compounds 13–19 (iso-butyric group at the N-terminal of the GPA tripeptide), the presence of allyl, dimethylallyl, and cinnamyl groups was best tolerated by BACE-1 as compounds 15, 16, and 17 exhibited IC₅₀ values of 0.05, 0.2, and 0.1 μM, respectively (Table 1). In the case of compounds 21–27 (Cbz group at the N-terminal of GPA), compounds 25, 26, and 27 with cinnamyl, geranyl, and crotyl groups, respectively, at the O-terminus were found more potent. The IC₅₀ values of compounds 25, 27, and 30 were similar to Calbiochem IV, which was employed as a control (IC₅₀ 20 nM). In general, it was observed that the presence of the substituents at both the N- and O-termini of the GPA tripeptide influences the BACE-1 inhibitory activities of the compounds.

Table 1. BACE-1 and AChE Inhibitory Activities of Proline and Binol Derivatives.

		IC50 (μM)
compound	structure	BACE-1	AChE	% inhibition of Aβ1–42 aggregation at 10 μM
13	isobut-GPA-OMe	0.2 ± 0.03	7 ± 1.0	36.3 ± 4.3
14	isobut-GPA–OH	0.6 ± 0.05	8 ± 1.2
15	isobut-GPA-O-allyl	0.05 ± 0.001	0.05 ± 0.01	51 ± 6.1
16	isobut-GPA-O-dimethylallyl	0.2 ± 0.01	1 ± 0.1
17	isobut-GPA-O-cinnamyl	0.1 ± 0.01	0.9 ± 0.1
18	isobut-GPA-O-geranyl	20 ± 2.0	7 ± 1.3	12.48 ± 2.1
19	isobut-GPA-O-crotyl	30 ± 3.5	5 ± 1.1
21	Cbz-GPA-OMe	30 ± 4.1	1000 ± 9.5
22	Cbz-GPA–OH	0.3 ± 0.02	0.1 ± 0.01
23	Cbz-GPA-O-allyl	1 ± 0.2	0.8 ± 0.1
24	Cbz-GPA-O-dimethylallyl	20 ± 2.3	0.6 ± 0.03
25	Cbz-GPA-O-cinnamyl	0.02 ± 0.001	1 ± 0.2	54.27 ± 8.4
26	Cbz-GPA-O-geranyl	0.1 ± 0.01	0.7 ± 0.1	31.79 ± 3.2
27	Cbz-GPA-O-crotyl	0.01 ± 0.001	1 ± 0.2
30	NH2-GPA-OMe	0.02 ± 0.001	2 ± 1.1	33.71 ± 4.2
33	binol-O-cinnamyl azole	1 ± 0.1	7 ± 1.4	43.22 ± 4.7
	donepezil		0.09 ± 0.01	10.95 ± 1.4
	calbiochem	0.02 ± 0.001

2.3.2. AChE Inhibitory Activities

All of the test compounds except for 15 were found to have IC₅₀ values in the micromolar range. Compound 15 displayed an IC₅₀ of 0.05 μM (Table 1), which was even better than the standard drug donepezil (IC₅₀ 0.09 μM). A good correlation between the potency of the compounds and their extent of interactions with AChE was observed. The molecular docking of compound 15 (Figure 3) and its homologue 23 (Figure S10) in AChE showed that compound 23 is slightly displaced from the active site pocket. The higher IC₅₀ of compound 16 in comparison to compound 15 may be attributed to its small number of interactions with the enzyme as was evident from the molecular docking studies. Compound 16 showed only one H-bond interaction with the water molecule present in the active site of the enzyme (Figure S4). Among the N-Cbz compounds 21–27, compound 22 exhibited an IC₅₀ value of 0.1 μM. The better activity of compound 22 also appeared due to its H-bond and π–π interactions with the amino acid residues of AChE (Figure S9). On the other hand, its methyl derivative 21 with IC₅₀ in the millimolar range did not interact with any of the amino acids present in the active site of AChE. Compounds 13 and 14 showed IC₅₀ values in the micromolar range, and they were found to exhibit a H-bond interaction with water and π–π stacking with H447, respectively (Figures S1 and S2). Derivatization of the free acid functionality of compound 14 with crotyl, geranyl, cinnamyl, dimethyl, and allyl substituents resulted in better IC₅₀ values, whereas derivatization of 22 with these substituents increased the inhibitory concentrations of the compounds. In accordance with the results of molecular docking studies, the benzyl derivative 27 showed better inhibitory activity in comparison to compound 19. Compound 33 also exhibited activity against BACE-1 and AChE although the presence of the binol moiety did not appear, providing a major additional advantage to the molecules.

2.3.3. Inhibition of Aβ_1–42 Aggregation

Irrespective of the influence of metal ions, AChE, and oxidative stress, the aggregation of Aβ is unclear; here, the compounds 13, 15, 18, 25, 26, 27, 30, and 33 were investigated for their ability to inhibit Aβ_1–42 aggregation. Compound 15, the potent inhibitor of both AChE (IC₅₀ 0.05 μM) and BACE-1 (IC₅₀ 0.05 μM), was found to inhibit Aβ_1–42 aggregation by 51%, while another potent compound 25 exhibited 54% inhibition of Aβ_1–42 aggregation (Figure 4).

Figure 4.

Percentage inhibition of Aβ_1–42 aggregation at 10 μM exhibited by compounds 13, 15, 18, 25, 26, 30, and 33.

2.3.4. Bioavailability

Penetration through the biological membranes is a critical aspect of the CNS targeting drugs. Compounds 15, 17, 18, 19, 25, 27, 30, and 33 were examined for their ability to passively move in the parallel artificial membrane permeability experiment (Figure 5). Significant permeability of all of the compounds was observed with Pe values exceeding the breakpoint for permeable compounds given by PAMPA plate’s manufacturer, i.e., Pe 1.5 × 10^–6 cm s^–1. Generally, the CNS + compounds have Pe values >4 × 10^–6 cm s^–1, while CNS- exhibit Pe < 2 × 10^–6 cm s^–1. Pe values for compounds 19 and 25 were higher than the Pe values for the permeable compound caffeine (Pe = 10.9 × 10^–6 cm s^–1) (Table 2).

Figure 5.

Pictorial representation of the two plates of the PAMPA system mimicking the BBB.

Table 2. Permeability Values (Pe) Calculated from the PAMPA-BBB Assay.

compound	structure	Pe (cm s–1)
15	iso-GPA-O-allyl	1.80 ± 0.2 × 10–6
17	iso-GPA-O-cinnamyl	3.64 ± 0.7 × 10–6
18	iso-GPA-O-geranyl	3.17 ± 0.6 × 10–6
19	iso-GPA-O-crotyl	20.7 ± 3.5 × 10–6
25	Cbz-GPA-O-cinnamyl	17.8 ± 2.8 × 10–6
27	Cbz-GPA-O-crotyl	6.93 ± 1.3 × 10–6
30	NH2-GPA-OMe	8.98 ± 2.1 × 10–6
33	binol-O-cinnamyl azole	1.59 ± 0.3 × 10–6
	caffeinea	10.9 ± 1.9 × 10–6
	norfloxacinb	1.73 ± 0.2 × 10–6

^aLit.:³⁷ 2.7 × 10^–6 cm s^–1.

^b0.4 × 10^–6 cm s^–1 with lipid conc 100 mg mL^–1.

2.3.5. In Vivo Studies on Mice Models

2.3.5.1. Effect of Test Compounds on Latency Time: Time Taken by the Animal to Reach the Platform in the Morris Water Maze (MWM) Test

Animals were trained for 5 days so that they learn to reach the platform. The basal reading on the fifth day showed that all of the animals were trained adequately to be used in this study (Table 3). No significant difference in the latency time to reach the platform was observed in the different groups of animals (Figure 6). On the sixth day, the animal groups 3–8 were treated with donepezil (3 mg/kg), compound 30 (10 mg/kg), compound 15 (10 mg/kg), compound 18 (10 mg/kg), compound 25 (10 mg/kg), and compound 26 (10 mg/kg), respectively. On the 11th day (before scopolamine treatment), no significant difference in the time to reach the platform was observed in all of the animal groups except the animals of group 8 (compound 26 treated) (Figure 6A). Scopolamine treatment on the 12th, 13th, and 14th days increased the latency to reach the platform in comparison to the normal animals, thus indicating the memory-impairing effect of scopolamine (Table 3). Considerable reduction in the time taken by the animals in group 3 (donepezil-treated) to reach the platform was observed in comparison to the scopolamine-treated group 2 (Figure 6C). Noticeably, the pretreatment with the test molecules, compound 30 (10 mg/kg), compound 25 (10 mg/kg), and compound 26 (10 mg/kg) also alleviated the memory-impairing effect of scopolamine when a significant reduction in the latency time to reach the platform was observed in animal groups 4, 7, and 8. However, pretreatment with compounds 15 and 18 did not show a significant effect on scopolamine-induced memory impairment. Compound 15, although highly potent in the in vitro experiments, had a poor effect on scopolamine-induced memory impairment, which may be attributed to its low permeability through the biological membrane. The effect of compound 25 was similar to that observed with the standard drug donepezil (Figure 6C and Table 4).

Table 3. Starting from the Blue Dot, Trajectory Plots of Each Animal from the Different Groups.

Figure 6.

(A) Basal reading on day 5 after 4 days of training and before the start of the test treatment in MWM. (B) Effect of various compounds (10 mg/kg) on the animals’ memory before scopolamine treatment. (C) Effect of the compounds (10 mg/kg) on the animals’ memory after inducing impaired memory with scopolamine. Data were analyzed by using a one-way analysis of variance followed by Tukey’s test (^ap < 0.05 vs the control group, ^bp < 0.05 vs scopolamine).

Table 4. Reversal of Scopolamine-Induced Memory Impairment in the Presence of the Test Compound. This Data Corresponds to One Animal from Each Group.

	time (in s) taken by the animals to reach the platform
groups	after the training (5th day)	after treatment with the test compound/donepezil (11th day)	after treatment with scopolamine (14th day)
control	6	6	10
scopolamine	8	3	77
donepezil	15	17	10
15	11	11	93
18	5	5	71
25	20	15	15
26	7	39	45
30	20	12	65

2.3.5.2. Effect of the Test Compounds on the Latency Time to Reach the Shock-Free Area in the Passive Avoidance Task (PAT)

As observed in the MWM test, no significant difference in the latency time to reach the shock-free area was seen in the PAT test after the standard and the test treatments (until the 11th day, before scopolamine treatment) (Figure 7A). Scopolamine treatment for the three consecutive days (12th, 13th, and 14th days) significantly increased the latency time, indicating its memory-impairing effect. Pretreatment with donepezil (3 mg/kg) reduced the latency to reach the shock-free area in a short while as compared to the scopolamine-treated group. Similar to donepezil, pretreatment with compound 30 (10 mg/kg), compound 18 (10 mg/kg), compound 25 (10 mg/kg), and compound 26 (10 mg/kg) also improved the memory-impairing effect of scopolamine. However, pretreatment with compound 15 did not have a significant effect on scopolamine-induced memory impairment in the PAT test too (Figure 7B).

Figure 7.

PAT assay: effect of the compounds (10 mg/kg) on memory in PAT (A) before scopolamine treatment and (B) after treatment with scopolamine to induce impaired memory in PAT. Data were analyzed by using a one-way analysis of variance followed by Tukey’s test (^ap < 0.05 vs the control group, ^bp < 0.05 vs scopolamine).

2.3.6. Lipinski Parameters and ADME Profile

Drug likeness properties such as Lipinski parameters (Table 5) and the ADME profile (Table S2) of the test compounds were checked and correlated with the results of biological studies. Molecular weight of all of the compounds is <500 Da except for compound 33. It has been proposed that compounds with high lipophilicity (c log P > 3) are likely to have a higher chance of toxicity in animal studies in comparison to compounds with lower lipophilicity (c log P < 3).³⁸ Most of the compounds were found to exhibit c log P < 3 except for compounds 18, 26, 32, and 33. Since donepezil was found to be extensively metabolized by CYP 2D6 and CYP 3A4 isoforms of CYP 450, we also estimated the possibility of metabolism of the designed compounds with these two isoforms using PreADME software. Only compound 30 was found to be a weak substrate of CYP 2D6 among the series (Table S2). Compounds 12, 13, 19, and 21–29 appeared as the substrates of CYP 3A4, whereas the binol derivatives (compounds 33, 32) displayed an enzyme inhibition effect. When given orally, a compound with higher human intestinal absorption (HIA) is more likely to be absorbed from the intestinal tract. Except for compound 31, all of the other compounds had HIA > 50. Plasma protein binding can also enhance the solubility of the molecules, especially the hydrophobic ones, as well as it protects the drugs from oxidation, reduces toxicity, and extends their half-life. All of the compounds were found to have good plasma binding capacity.

Table 5. Lipinski Parameters of the Compounds^a.

compound	mi log P	TPSA (Å2)	MW (g/mol)	nON	nOHNH
1	4.27	73.22	409.44	5	0
2	6.60	38.78	552.48	4	0
3	6.50	38.78	534.49	4	0
4	4.62	74.60	294.39	4	2
5	2.68	90.89	270.24	5	3
6	3.30	15.27	302.37	2	1
7	5.35	135.73	641.68	12	2
8	4.74	103.97	608.74	9	2
13	–0.42	104.81	327.38	8	2
14	–2.49	115.80	313.25	8	3
20	–0.44	66.40	193.20	4	2
21	0.26	104.81	375.43	8	2
22	–1.81	115.80	361.40	8	3
15	0.23	104.81	353.42	8	2
16	1.26	104.81	381.47	8	2
17	1.93	104.81	429.52	8	2
18	3.10	104.81	449.59	8	2
19	0.34	131.12	425.48	10	2
23	0.91	104.81	401.46	8	2
24	1.94	104.81	429.52	8	2
25	2.61	104.81	477.56	8	2
26	3.78	104.81	497.64	8	2
27	1.02	131.12	473.53	10	2
28	–0.42	58.56	147.17	4	2
29	0.28	96.97	329.40	8	2
30	–1.84	101.73	257.29	7	3
32	6.39	18.47	362.43	2	0
33	8.93	79.90	680.81	8	0

^a1–8: Reported compounds (Figure 2A); 9–33: compounds of present investigations.

2.3.7. Plasma Stability Assay

Compound 25 was checked for its stability through a plasma stability assay.³⁹ A solution of compound 25 in PBS (50 μM) was used as the positive control. Compound 25 was taken in plasma (pH 7–8) to produce the solution of final concentration 50 μM. After incubation of the sample at 37 °C for 1 h and 12 h, 400 μL of acetonitrile was added to quench the reaction. The concentration of the compound in plasma was quantified from the HRMS chromatograms of the treated supernatant and buffer solutions. The percentage of analyte lysed by plasma of each solution was calculated from intensities of peaks observed in the HRMS spectra at the same interval of time using eq 1.

1

where I₀ and I represent the intensities of compounds in the presence of buffer and plasma (treated supernatant), respectively. Remarkably, a slight change in the concentration of 25 (∼10%) was observed after 12 h of incubation (Figures S137–S139). It is demonstrated that plasma had no remarkable effect on the half-life time of 25, and further, it can be prioritized for in vivo studies.

3. Conclusions and Remarks

The compounds designed after analyzing the catalytic modes of BACE-1 and AChE proved highly effective against the Alzheimer’s disease animal models. The functioning of the catalytic processes of these two enzymes through the participation of a polar diad consisting of Asp228 and Asp32 for BACE-1 and the triad of Glu334, His447, and Ser203 for AChE laid the basis for the design of new molecules having a higher total polar surface area (TPSA) than the reported compounds (1–8, Table 5) so that they better interact in the catalytic pocket of the enzymes. The derivatization of the Gly–Pro–Ala tripeptide through its N- and O-termini was appropriately achieved in making the required hydrophobic–hydrophilic balance in the molecule (logP and TPSA). In comparison to compounds 1–8 in Table 5, compound 25 has log P 2.61 and TPSA 104. The in vitro and in vivo screening of the molecules led to the identification of highly potent molecules. While appreciably potent in the enzyme immunoassays, compound 25 exhibited the best biological membrane permeability, and it was identified as the most effective in reversing scopolamine-induced memory impairment in mice models. It implies that a suitable combination of log P and TPSA is desirable for the better biological activity of the molecule. In accordance with the reported observations, it was noticed that a compound with considerable in vitro potency may not be effective under the in vivo conditions; such a case was represented by compound 15. Therefore, adopting a rational approach of drug designing, the integration of in silico and wet-lab experiments resulted in the identification of some highly potent molecules, which can be taken to the next level of investigations.

4. Experimental Section

4.1. General

All of the solvents were of chromatography grade and used without further purification. The chemicals were purchased either from Sigma-Aldrich or from S.d. fine/Spectrochem. Thin-layer chromatography (TLC) was performed using silica plates precoated with silica gel GF-254 (Qualigens, India). Column chromatography was performed with a silica 60–120 mesh. LC-MS and high-resolution mass spectra were recorded on a Bruker micrOTOF-QII mass spectrometer using the +ve/–ve ion mode. Melting points were determined in capillaries. ¹H and ¹³C NMR spectra were recorded in CDCl₃ and/or DMSO-d₆ on a Bruker 500 and 125 MHz NMR spectrometer, respectively. Chemical shifts are given in ppm with TMS as an internal reference. Coupling constants (J) are given in Hz. Signals are abbreviated as singlet, s; doublet, d; double–doublet, dd; triplet, t; and multiplet, m. In ¹³C/DEPT-135 data, the +ve sign corresponds to signals due to CH₃ and CH groups, while the −ve sign symbolizes signals of CH₂ groups, and signals of quaternary carbon are absent (ab) in the DEPT-135 spectra. IR and UV–vis spectral data were recorded on FTIR Agilent CARY 630 and BIOTEK Synergy H1 Hybrid Reader instruments, respectively. Scopolamine was purchased from Sovereign Pharma Pvt. Ltd., India. Donepezil was purchased from Sigma-Aldrich. Percentage purity of the compounds was checked with q¹H NMR spectra.⁴⁰

4.2. General Synthetic Procedure

4.2.1. Procedure A

A suspension of l-amino acid (0.01 mol) in 10 mL of dioxane/water (2:1) was dissolved in 0.01 mol sodium hydroxide at room temperature (1 M). After stirring the reaction mixture for 5 min at room temperature, 0.01 mol of Boc₂O was added and continuously stirred for 1.5 h. The dioxane was then vacuum-evaporated, and the aqueous portion was acidified to pH 2 using dil HCl. To obtain the required N-Boc-AA–OH, the released oil was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under vacuum.

4.2.2. Procedure B

l-Amino acid (0.01 mol) was dissolved in 4 M sodium hydroxide solution (0.01 mol) and chilled to 0 °C. Over 30 min, 0.01 mol of benzyl chloroformate and 0.01 mol of 4 M sodium hydroxide solution were added alternatively and portion-wise while stirring at 0 °C. The reaction mixture was then allowed to stir for 1 h at room temperature. The reaction mixture’s contents were rinsed with 2 × 15 mL of ether, acidified with dil HCl to pH 2, extracted with ether, dried over anhydrous sodium sulfate, and concentrated in vacuum.

4.2.3. Procedure C

Triethylamine (0.021 mol) was added to a solution of N-Boc-AA (N-Boc-amino acid) (1 mmol) in THF (50 mL) at −10 °C followed by the addition of ethyl chloroformate (0.014 mol). To this reaction mixture, neutralized AA methyl ester hydrochloride (1.2 mmol) in 5 mL of THF was added at −10 °C. The reaction mixture was stirred at 0 °C to −10 °C for 1 h and then shifted to stir at 25 ± 2 °C for 12 h. After completion of the reaction, THF was evaporated under vacuum. The residue was then dissolved in 100 mL of ethyl acetate and washed with water (30 mL). The organic layer was washed with 5% Na₂CO₃ solution and 5% citric acid solution, dried over anhydrous Na₂SO₄, and evaporated to dryness under vacuum. The crude product was purified with column chromatography using ethyl acetate:hexane as the eluent to yield pure N-Boc-AA-AA methyl ester.

4.2.4. Procedure D

A solution of N-Boc-AA-AA methyl ester (0.01 mol) in acetone–water (3:2) was progressively added to 1 N NaOH (0.01 mol) at 25 ± 2 °C. The reaction mixture was stirred for 1 h. After the reaction was complete, acetone was evaporated under vacuum. The remaining part of the reaction mixture was acidified with dil HCl, and the product was extracted with ethyl acetate.

4.2.5. Procedure E

N-Boc-AA-AA(OMe) in dichloromethane (DCM) was added to a solution of trifluoroacetic acid (10 equiv of the peptide) and stirred at room temperature for 8 h. After completion of the reaction (TLC), the reaction mixture was concentrated in vacuum followed by washing with diethyl ether (3 × 10 mL) to obtain the desired product.

4.2.6. Procedure F

To the solution of AA–OH (1 mmol) in ACN at 60 °C, K₂CO₃ (1.5 mmol) was added, followed by the addition of allyl bromide/dimethyl bromide/crotyl bromide/cinnamyl bromide/geranyl bromide (1.2 mmol). The reaction was stirred at 60 °C for 12 h. After quenching the reaction mixture with ice, the crude product was extracted with ethyl acetate, followed by washing with water (30 mL). After drying over anhydrous sodium sulfate, the excess solvent was evaporated under vacuum to get the desired compound.

4.2.7. Procedure G

(S)-BINOL (1 mmol) in acetone was taken in a round-bottom flask fixed with a reflux condenser. To this solution, anhydrous potassium carbonate (3 mmol) was added, followed by the addition of propargyl bromide (3 mmol). The reaction mixture was allowed to stir for 16 h at 60 °C. The reaction mixture was evaporated on a rotary evaporator. The residue was purified by recrystallization (ethyl acetate/diethyl ether) to give compound 31 in 95% yield.

4.2.8. Procedure H

To the solution of cinnamyl bromide (1 mmol) in DMF, sodium azide (3 mmol) was added. The reaction mixture was allowed to stir at 60 °C for 2 h. After the completion of reaction, it was quenched with water and extracted with ethyl acetate. Then, the ethyl acetate layer was passed through Na₂SO₄ and the solvent was evaporated on a rotary evaporator to get the compound as a pale-yellow liquid.

4.2.9. Procedure I

To the solution of compound 31 (0.35 mmol) in ethanol (7 mL), sodium ascorbate (0.01 mmol) and CuSO₄.5H₂O (0.03 mmol) were added. Further, a solution of cinnamyl azide (1.78 mmol) in ethanol was added to it. The reaction mixture was allowed to stir at room temperature for 12 h. After completion of the reaction (TLC), the reaction mixture was extracted with ethyl acetate. Ethyl acetate was then evaporated on a rotary evaporator to get a thick oil as the desired product.

4.2.10. Boc-Pro-OH (9)

Compound 9 was synthesized according to the general procedure A as a colorless liquid; yield 90%; HRMS (microTOF-QII, MS, ESI): calcd for C₁₀H₁₇O₃N ([M + H]⁺) 222.1100, found 222.1103.

4.2.11. Boc-Pro-Ala-OMe (10)

Compound 10 was synthesized according to the general procedure C as a colorless liquid; yield 40%; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.40 (d, J = 6.56 Hz, 3H, CH_3Ala), 1.48 (s, 9H, CH_3Boc), 1.84–2.02 (m, 2H, CH_2Pro), 2.05–2.32 (m, 2H, CH_2Pro), 3.36–3.50 (m, 2H, CH_2Pro), 3.75 (s, 3H, OCH₃), 4.08–4.31 (m, 1H, CH_Ala), 4.49–4.64 (m, 1H, CH_Pro), 7.28 (br, 1H, NH); HRMS (microTOF-QII, MS, ESI): calcd for C₁₄H₂₄O₅N₂ ([M + Na]⁺) 323.1577, found 323.1579.

4.2.12. NH-Pro-Ala-OMe (11)

Compound 11 was synthesized using the general procedure E as a colorless liquid; yield 40%; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.33 (d, J = 6.56 Hz, 3H, CH_3Ala), 1.76–1.99 (m, 3H, CH_2Pro), 2.31–2.34 (m, 1H, CH_2Pro), 3.20–3.24 (m, 2H, CH_2Pro), 3.64 (s, 3H, OCH₃), 4.24–4.25 (m, 1H, CH_Ala), 4.32–4.35 (m, 1H, CH_Pro), 8.88 (d, J = 6.88 Hz, 1H, NH), 9.02 (d, J = 6.18 Hz, 1H, NH); HRMS (microTOF-QII, MS, ESI): calcd for C₉H₁₆O₃N₂ ([M + H]⁺) 201.1233, found 201.1282.

4.2.13. Isobut-Gly-OH (12)

Compound 12 was synthesized according to the general procedure D as a colorless liquid; yield 40%, mp 107 °C; ¹H NMR (500 MHz, DMSO-d₆, 25 °C, TMS): δ 1.01 (d, J = 7.08 Hz, 6H, CH₃), 2.39–2.44 (m, 1H, CH), 3.71 (d, J = 6.03 Hz, 2H, CH_2Gly), 8.06 (t, J = 5.58 Hz, 1H, CH_NH), 12.45 (br, 1H, OH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 19.88 (+ve, CH₃), 34.18 (+ve, CH), 40.90 (−ve, CH₂), 43.46 (−ve, CH_2Gly), 171.95 (C=O), 176.94 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₆H₁₁O₃N ([M + Na]⁺) 168.0631, found 168.0657.

4.2.14. Isobut-Gly-Pro-Ala-OMe (13)

Compound 13 was synthesized according to the general procedure C as a white solid, yield 76%, mp 172 °C; [α]²⁵_D −105° (0.4, CHCl₃); IR (ATR): 3257, 3086, 2959, 2363, 1729, 1640, 1558, 1453, 1237, 1051, 998, 924, 775, 685 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.17 (d, J = 6.97 Hz, 6H, 2 x CH_Iso), 1.40 (t, 3H, J = 7.13 Hz, CH_3Ala), 1.94–2.04 (m, 2H, CH_2Pro), 2.12–2.17 (m, 1H, CH_2Pro), 2.29–2.34 (m, 1H, CH_2Pro), 2.42–2.50 (m, 1H, CH_Iso), 3.45 (q, J = 9.70 Hz, 1H, CH), 3.56–3.61 (m, 1H, CH_2Pro), 3.74 (s, 3H, OCH₃), 3.97–4.01 (m, 1H, CH), 4.09–4.13 (m, 1H, CH), 4.48–4.55 (m, 2H, OCH₂), 6.51 (br, 1H, NH), 7.04 (d, J = 6.94 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.07 (+ve, CH₃), 19.49 (+ve, CH₃), 19.55 (+ve, CH₃), 24.79 (−ve, CH_2Pro), 27.95 (−ve, CH_2Pro), 35.36 (+ve, CH), 42.01 (−ve, CH_2Gly), 46.47 (−ve, CH_2Pro), 48.24 (+ve, CH_Ala), 52.46 (+ve, CH_Pro), 60.14 (−ve, CH₂), 168.30 (C=O), 170.49 (C=O), 172.29 (C=O), 177.11 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₅H₂₅O₅N₃ ([M + Na]⁺) 350.1686, found 350.1674.

4.2.15. Isobut-Gly-Pro-Ala-OH (14)

Compound 14 was synthesized according to the general procedure D as a white solid, yield 76%, mp 184 °C; [α]²⁵_D −102.5° (0.4, CHCl₃); IR (ATR): 3295, 3220, 3071, 2974, 2363, 1729, 1640, 1550, 1453, 1222, 1148, 1058, 924, 857, 775 cm^–1; ¹H NMR (500 MHz, DMSO-d₆, 25 °C, TMS): δ 1.00 (d, J = 6.76 Hz, 6H, 2 x CH_Iso), 1.27 (m, 3H, J = 7.13 Hz, CH_3Ala), 1.75–1.78 (m, 1H, CH_2Pro), 1.83–1.91 (m, 2H, CH_2Pro), 1.97–2.04 (m, 1H, CH_2Pro), 2.43–2.48 (m, 1H, CH_Iso), 3.43–3.48 (m, 1H, CH_2Pro), 3.50–3.55 (m, 1H, CH_2Pro), 3.75–3.79 (m, 1H, CH_2Gly), 3.93–3.97 (m, 1H, CH_2Gly), 4.11–4.17 (m, 1H, CH), 4.33–4.35 (m, 1H, CH), 7.82–7.86 (m, 1H, NH), 6.51 (br, 1H, NH), 8.18 (d, J = 7.36 Hz, 1H, NH), 12.56 (br, 1H, OH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 17.50 (+ve, CH₃), 19.98 (+ve, CH₃), 20.03 (+ve, CH₃), 24.61 (−ve, CH_2Pro), 29.49 (−ve, CH_2Pro), 34.14 (+ve, CH), 41.43 (−ve, CH_2Gly), 46.28 (−ve, CH_2Pro), 47.85 (+ve, CH_Ala), 48.04 (+ve, CH_Pro), 59.55 (−ve, CH₂), 167.52 (C=O), 171.87 (C=O), 174.51 (C=O), 176.74 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₄H₂₃O₅N₃ ([M + K]⁺) 352.1265, found 352.1269.

4.2.16. Isobut-Gly-Pro-Ala-O-allyl (15)

Compound 15 was synthesized according to the general procedure F as a white solid, yield 70%, mp 150 °C; [α]²⁵_D −40° (0.4, CHCl₃); IR (ATR): 3257, 3086, 2974, 2087, 1744, 1640, 1558, 1453, 1326, 1200, 158, 924, 693, 625, 603 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.17 (d, J = 6.94 Hz, 6H, 2 x CH_Iso), 1.42 (t, 3H, J = 7.03 Hz, CH_3Ala), 1.92–2.04 (m, 2H, CH_2Pro), 2.11–2.19 (m, 1H, CH_2Pro), 2.30–2.34 (m, 1H, CH_2Pro), 2.41–2.48 (m, 1H, CH_Iso), 3.45 (q, J = 9.72 Hz, 1H, CH), 3.56–3.60 (m, 1H, CH_2Pro), 3.97–4.01 (m, 1H, CH), 4.07–4.13 (m, 1H, CH), 4.50–4.56 (m, 2H, CH₂), 4.61–4.66 (m, 2H, CH₂), 5.25 (dd, J = 10.65 Hz, 1.23 Hz, 1H,=CH), 5.33 (dd, J = 17.25 Hz, 1.49 Hz, 1H, = CH), 5.87–5.94 (, 1H, CH), 6.48 (br, 1H, NH), 7.00 (d, J = 7.02 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.11 (+ve, CH₃), 19.49 (+ve, CH₃), 19.56 (+ve, CH₃), 24.80 (−ve, CH_2Pro), 27.90 (−ve, CH_2Pro), 35.39 (+ve, CH), 42.02 (−ve, CH_2Gly), 46.47 (−ve, CH_2Pro), 48.36 (+ve, CH_Ala), 60.16 (+ve, CH_Pro), 65.95 (−ve, CH₂), 118.72 (−ve, = CH₂), 131.57 (+ve, = CH), 168.31 (C=O), 170.44 (C=O), 172.47 (C=O), 177.09 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₇H₂₇O₅N₃ ([M + Na]⁺) 376.1842, found 376.1850.

4.2.17. Isobut-Gly-Pro-Ala-O-dimethylallyl (16)

Compound 16 was synthesized according to the general procedure F as a white solid, yield 73%, mp 149 °C; [α]²⁵_D −87.5° (0.4, CHCl₃); IR (ATR): 3257, 3078, 2974, 2124, 1729, 1632, 1558, 1453, 1237, 1043, 948, 685 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.17 (d, J = 6.88 Hz, 6H, 2 x CH_Iso), 1.40 (t, 3H, J = 7.15 Hz, CH_3Ala), 1.71 (s, 3H, CH₃), 1.76 (s, 1H, CH₃), 1.94–2.03 (m, 2H, CH_2Pro), 2.11–2.19 (m, 1H, CH_2Pro), 2.29–2.33 (m, 1H, CH_2Pro), 2.41–2.48 (m, 1H, CH), 3.42–3.47 (m, 1H, CH_2Pro), 3.56–3.60 (m, 1H, CH_2Pro), 3.96–4.00 (m, 1H, CH), 4.10–4.14 (m, 1H, CH), 447–4.53 (m, 2H, CH₂), 4.59–4.66 (m, 2H, CH₂), 5.31–5.34 (m, 1H, = CH), 6.51 (br, 1H, NH), 6.97 (d, J = 7.04 Hz, 1H, CH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.08 (+ve, CH₃), 18.22 (+ve, CH₃), 19.50 (+ve, CH₃), 19.56 (+ve, CH₃), 24.79 (−ve, CH_2Pro), 25.79 (+ve, CH₃), 28.02 (−ve, CH_2Pro), 35.38 (+ve, CH), 42.02 (−ve, CH_2Gly), 46.45 (−ve, CH_2Pro), 48.38 (+ve, CH_Ala), 60.19 (+ve, CH_Pro), 62.34 (−ve, CH₂), 118.01 (+ve, = CH), 139.81 (C), 168.23 (C=O), 170.41 (C=O), 172.84 (C=O), 177.10 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₉H₃₁O₅N₃ ([M + Na]⁺) 404.2155, found 404.2156.

4.2.18. Isobut-Gly-Pro-Ala-O-Cinnamyl (17)

Compound 17 was synthesized according to the general procedure F as a white solid, yield 76%, mp 158 °C; [α]²⁵_D −95° (0.4, CHCl₃); IR (ATR): 3257, 3086, 2959, 2877, 2363, 2109, 1714, 1632, 1453, 1380, 1237, 1103, 984, 693, 521 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.17 (d, J = 6.85 Hz, 6H, 2 × CH_Iso), 1.44 (t, 3H, J = 7.18 Hz, CH_3Ala), 1.90–2.03 (m, 2H, CH_2Pro), 2.11–2.17 (m, 1H, CH_2Pro), 2.30–2.35 (m, 1H, CH_2Pro), 2.40–2.48 (m, 1H, CH_Iso), 3.43–3.45 (m, 1H, CH_2Pro), 3.56–3.60 (m, 1H, CH_2Pro), 3.96–3.97 (m, 1H, CH), 4.12–4.13 (m, 1H, CH), 4.55 (q, J = 7.28 Hz, 2H, OCH₂), 4.76–4.83 (m, 2H, CH₂), 6.24–6.29 (m, 1H, = CH), 6.48 (br, 1H, NH), 6.65–6.68 (m, 1H, = CH), 7.02–7.05 (m, 1H, NH), 7.27–7.28 (m, 1H, ArCH), 7.31–7.34 (m, 2H, ArCH), 7.38–7.40 (m, 2H, ArCH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.14 (+ve, CH₃), 19.45 (+ve, CH₃), 19.56 (+ve, CH₃), 24.79 (−ve, CH_2Pro), 27.92 (−ve, CH_2Pro), 35.38 (+ve, CH), 42.03 (−ve, CH_2Gly), 46.47 (−ve,CH_2Pro), 48.42 (+ve, CH_Ala), 60.17 (+ve, CH_Pro), 65.99 (−ve, CH₂), 122.46 (+ve, ArCH), 126.67 (+ve, ArCH), 128.65 (+ve, ArCH), 134.73 (+ve, CH), 136.02 (ArC), 168.31 (C=O), 170.47 (C=O), 172.60 (C=O), 177.12 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₃H₃₁O₅N₃ ([M + Na]⁺) 452.2155, found 452.2165.

4.2.19. Isobut-Gly-Pro-Ala-O-Geranyl (18)

Compound 18 was synthesized according to the general procedure F as a white solid, yield 81%, mp 125 °C; [α]²⁵_D −50° (0.4, CHCl₃); IR (ATR): 3265, 3086, 2974, 2363, 2102, 1744, 1632, 1558, 1453, 1386, 1237, 1051, 946, 685, 603 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.16–1.18 (m, 6H, 2 × CH_Iso), 1.39–1.41 (m, 3H, CH₃), 1.58–1.62 (m, 3H, CH₃), 1.67–1.70 (m, 6H, 2 × CH₃), 1.91–2.09 (m, 6H, 2 × CH_2Ger), 2.15–2.19 (m, 1H, CH_2Pro), 2.24–2.30 (m, 1H, CH_2Pro), 2.41–2.50 (m, 1H, CH_Iso), 3.44 (q, J = 9.10 Hz, 1H, CH_2Pro), 2.43–2.49 (m, 1H, CH_2Iso), 3.45 (q, J = 9.56 Hz, 1H, CH_2Pro), 3.56–3.60 (m, 1H, CH_2Pro), 3.96–4.00 (m, 1H, CH), 4.10–4.15 (m, 1H, CH), 4.48–4.53 (m, 2H, CH₂), 4.63–4.66 (, 2H, CH₂), 5.07 (t, J = 6.54 Hz, 1H, CH), 5.32 (t, J = 6.50 Hz, 1H, CH), 6.53 (br, 1H, NH), 7.03 (br, 1H, NH); DEPT-135 NMR (125 MHz, normal): δ 16.51 (+ve, CH₃), 17.71 (+ve, CH₃), 18.21 (+ve, CH₃), 19.50 (+ve, CH₃), 19.56 (+ve, CH₃), 24.78 (−ve, CH_2Pro), 25.70 (+ve, CH₃), 26.23 (−ve, CH_2Pro), 28.06 (−ve, CH₂), 32.00 (−ve, CH₂), 35.36 (+ve, CH), 39.50 (−ve, CH₂), 42.01 (−ve,CH₂), 46.45 (−ve, CH_2Pro), 48.36 (+ve, CH_Ala), 60.17 (+ve, CH_Pro), 62.80 (−ve, CH₂), 117.71 (+ve, CH), 123.62 (+ve, CH), 165.75 (C=O), 168.45 (C=O), 170.57 (C=O), 172.14 (C=O), 177.19 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₄H₃₉O₅N₃ ([M + H]⁺) 472.2781, found 472.2779.

4.2.20. Isobut-Gly-Pro-Ala-O-Crotyl (19)

Compound 19 was synthesized according to the general procedure F as a white solid, yield 80%, mp 136 °C; [α]²⁵_D −65° (0.4, CHCl₃); IR (ATR): 3257, 3086, 2981, 2124, 1722, 1632, 1558, 1453, 1244, 1148, 685, 484 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.17 (d, J = 6.93 Hz, 6H, 2 × CH_Iso), 1.30 (t, 3H, J = 7.40 Hz, CH_3Cro), 1.45 (d, J = 7.24 Hz, 3H, CH_3Ala), 1.92–1.98 (m, 1H, CH_2Pro), 2.00–2.05 (m, 1H, CH_2Pro), 2.09–2.17 (m, 1H, CH_2Pro), 2.32–2.36 (m, 1H, CH_2Pro), 2.43–2.49 (m, 1H, CH_2Pro), 3.45 (q, J = 9.56 Hz, 1H, CH), 3.57–3.61 (m, 1H, CH_2Pro), 3.97–4.02 (m, 1H, CH), 4.09–4.13 (m, 1H, CH), 4.19–4.23 (q, J = 7.12 Hz, 2H, OCH₂), 4.52–4.58 (m, 2H, CH₂), 4.76–4.84 (m, 2H, CH₂), 6.03–6.06 (m, 1H, CH), 6.50 (br, 1H, NH), 6.90–6.95 (m, 1H, CH), 7.13 (d, J = 7.36 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 14.23 (+ve, CH_Cro), 17.98 (+ve, CH_3Ala), 19.50 (+ve, CH_3Iso), 19.57 (+ve, CH_3Cro), 24.81 (−ve, CH_2Pro), 27.80 (−ve, CH_2Pro), 35.34 (+ve, CH), 42.02 (−ve, CH_2Gly), 46.52 (−ve, CH_2Pro), 48.33 (+ve, CH_Ala), 60.10 (+ve, CH_Pro), 60.72 (−ve, CH₂), 63.36 (−ve, OCH₂), 122.56 (+ve, CH), 140.40 (+ve, CH), 165.75 (C=O), 168.45 (C=O), 170.57 (C=O), 172.14 (C=O), 177.19 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₀H₃₁O₇N₃ ([M + Na]⁺) 448.2054, found 448.2087.

4.2.21. Cbz-Gly-OH (20)

Compound 20 was synthesized according to the general procedure A as a white solid, yield 80%; mp 119 °C; HRMS (microTOF-QII, MS, ESI): calcd for C₁₀H₁₁O₄N ([M + Na]⁺) 232.0580, found 232.0539.

4.2.22. Cbz-Gly-Pro-Ala-OMe (21)

Compound 21 was synthesized according to the general procedure C as a white solid, yield 75%, mp 129 °C; [α]²⁵_D −57.5° (0.4, CHCl₃); IR (ATR): 3309, 3085, 2944, 2363, 1714, 1550, 1453, 1237, 1155, 1058, 969, 879, 693, 462 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.38 (d, J = 7.15 Hz, 3H, CH_3Ala), 1.90–1.96 (m, 1H, CH_2Pro), 1.99–2.04 (m, 1H, CH_2Pro), 2.09–2.19 (m, 1H, CH_2Pro), 2.31–2.34 (m, 1H, CH_2Pro), 3.38–3.43 (m, 1H, CH_2Pro), 3.53–3.57 (m, 1H, CH_2Pro), 3.73 (s, 3H, OCH₃), 3.96–4.07 (m, 2H, CH_2Gly), 4.45–4.55 (m, 2H, CH_Ala + CH_Pro), 5.10–5.14 (m, 2H, CH₂), 5.71 (br, 1H, NH), 7.10 (d, J = 5.14 Hz, 1H, NH), 7.30–7.36 (m, 5H, ArH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.02 (+ve, CH₃), 24.86 (−ve, CH_2Pro), 27.66 (−ve, CH_2Pro), 43.46 (−ve, CH_2Gly), 46.38 (−ve,CH_2Pro), 48.27 (+ve, CH_Ala), 52.43 (+ve, CH_Pro), 60.12 (−ve, CH₂), 66.97 (−ve, CH₂), 128.05 (+ve, ArCH), 128.15 (+ve, ArCH), 128.52 (+ve, ArCH), 136.37 (ArC), 156.27 (C=O), 168.12 (C=O), 170.41 (C=O), 173.23 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₉H₂₅O₆N₃ ([M + H]⁺) 392.1816, found 392.1764.

4.2.23. Cbz-Gly-Pro-Ala-OH (22)

Compound 22 was synthesized according to the general procedure D as a white solid, yield 79%, mp 119 °C; [α]²⁵_D −62.5° (0.4, CHCl₃); IR (ATR): 3280, 3063, 2885, 2646, 2363, 2109, 1699, 1647, 1535, 1446, 1237, 1058, 984, 924, 775 cm^–1; ¹H NMR (500 MHz, DMSO-d₆, 25 °C, TMS): δ 1.25 (d, J = 7.24 Hz, 3H, CH_3Ala), 1.74–1.77 (m, 1H, CH_2Pro), 1.81–1.90 (m, 3H, CH_2Pro), 1.95–2.00 (m, 1H, CH_2Pro), 3.49–3.52 (m, 1H, CH_2Pro), 3.73–3.88 (m, 2H, CH_2Gly), 4.12–4.15 (m, 1H, CH_pro), 4.32–4.34 (m, 1H, CH_Ala), 5.01–5.03 (m, 2H, CH₂), 7.29–7.33 (m, 3H, CH_Ar), 7.35–7.36 (m, 3H, CH_Ar), 8.18 (d, J = 7.35 Hz, 1H, NH), 8.44 (d, J = 7.37 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 17.51 (+ve, CH_3Ala), 24.64 (−ve, CH_2Pro), 29.44 (−ve, CH_2Pro), 43.11 (−ve, CH_2Gly), 46.17 (−ve, CH_2Pro), 47.85 (+ve, CH_Ala), 59.55 (+ve, CH_Pro), 65.81 (−ve, CH₂), 128.14 (+ve, ArCH), 128.81 (+ve, ArCH), 137.58 (ArC), 156.93 (C=O), 167.50 (C=O), 171.87 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₉H₂₄O₆N₂ ([M + H]⁺) 378.1659, found 378.1623.

4.2.24. Cbz-Gly-Pro-Ala-O-Allyl (23)

Compound 23 was synthesized according to the general procedure F as a white solid, yield 79%, mp 92 °C; [α]²⁵_D −110° (0.4, CHCl₃); IR (ATR): 3309, 2858, 3063, 2952, 2363, 2109, 1744, 1647, 1535, 1446, 1326, 1252, 1155, 1058, 924, 834, 693, 603 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.40 (d, J = 7.18 Hz, 3H, CH_3Ala), 1.90–1.95 (m, 1H, CH_2Pro), 1.99–2.03 (m, 1H, CH_2Pro), 2.13–2.15 (m, 1H, CH_2Pro), 2.30–2.33 (m, 1H, CH_2Pro), 3.40–3.43 (m, 1H, CH_2Pro), 3.54–3.58 (m, 1H, CH_2Pro), 3.96–4.03 (m, 2H, CH_2Gly), 4.49–4.54 (m, 2H, CH_Ala + CH_Pro), 4.61–4.63 (m, 2H, OCH₂), 5.09–5.14 (m, 2H, CH₂), 5.24–5.34 (m, 2H, CH_2Allyl), 5.73 (br, IH, NH), 5.87–5.92 (m, 1H, CH_Allyl), 7.11 (br, 1H, NH), 7.31–7.33 (m, 5H, CH_Ar); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.03 (+ve, CH_3Ala), 24.86 (−ve, CH_2Pro), 27.75 (−ve, CH_2Pro), 43.46 (−ve, CH_2Gly), 46.40 (−ve, CH_2Pro), 47.26 (+ve, CH_Ala), 60.12 (+ve, CH_Pro), 65.93 (−ve, CH₂), 66.98 (−ve, CH_2Allyl), 118.71 (−ve, CH_2Allyl), 128.06 (+ve, ArCH), 128.16 (+ve, ArCH), 128.54 (+ve, ArCH), 131.59 (+ve, ArCH), 136.34 (ab, ArC), 156.28 (C=O), 168.11 (C=O), 170.49 (C=O), 172.47 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₁H₂₇O₆N₃ ([M + H]⁺) 440.1792, found 440.1768.

4.2.25. Cbz-Gly-Pro-Ala-O-dimethylallyl (24)

Compound 24 was synthesized according to the general procedure F as a brown thick oil, yield 83%; [α]²⁵_D −125° (0.4, CHCl₃); IR (ATR): 3354, 2929, 2832, 2363, 2102, 1662, 1513, 1297, 1237, 1036, 931, 827, 521 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.38 (t, 3H, J = 7.13 Hz, CH_3Ala), 1.70 (s, 3H, CH₃), 1.75 (s, 1H, CH₃), 1.91–1.94 (m, 1H, CH_2Pro), 1.97–2.01 (m, 1H, CH_2Pro), 2.11–2.15 (m, 1H, CH_2Pro), 2.30–2.32 (m, 1H, CH_2Pro), 3.38–3.43 (m, 1H, CH_2Pro), 3.54–3.57 (m, 1H, CH_2Pro), 3.95–4.07 (m, 2H, CH₂), 4.46–4.54 (m, 2H, CH_Ala + CH_Pro), 4.61–4.63 (m, 2H, OCH₂), 5.10–5.14 (m, 2H, CH₂), 5.31–5.33 (m, 1H, = CH), 5.73 (br, 1H, NH), 7.06 (m, J = 6.54 Hz, 1H, NH), 7.31–7.36 (m, 5H, ArH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.08 (+ve, CH₃), 18.15 (+ve, CH₃), 24.85 (−ve, CH_2Pro), 25.78 (+ve, CH₃), 27.79 (−ve, CH_2Pro), 43.46 (−ve, CH_2Gly), 46.37 (−ve, CH_2Pro), 48.40 (+ve, CH_Ala), 60.15 (+ve, CH_Pro), 62.30 (−ve, CH₂), 66.95 (−ve, CH₂), 118.05 (+ve, = CH), 128.04 (+ve, ArCH), 128.52 (+ve, ArCH), 136.37 (C) 139.75 (C), 156.26 (C=O), 168.02 (C=O), 170.37 (C=O), 172.79 (C=O), 177.10 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₃H₃₁O₆N₃ ([M + Na]⁺) 468.2105, found 468.2051.

4.2.26. Cbz-Gly-Pro-Ala-O-cinnamyl (25)

Compound 25 was synthesized according to the general procedure F as a brown thick oil, yield 82%; [α]²⁵_D −87.5° (0.4, CHCl₃); IR (ATR): 3295, 3056, 2959, 2885, 2363, 2109, 1997, 1744, 1640, 1535, 1453, 1244, 1148, 969, 834, 693, 603 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.41 (t, 3H, J = 7.20 Hz, CH_3Ala), 1.88–2.00 (m, 2H, CH_2Pro), 2.09–2.18 (m, 1H, CH_2Pro), 2.31–2.32 (m, 1H, CH_2Pro), 2.40–2.48 (m, 1H, CH_Iso), 3.37–3.42 (m, 1H, CH_2Pro), 3.53–3.56 (m, 1H, CH_2Pro), 3.95–4.06 (m, 2H, CH_Ala+Pro), 4.50–4.55 (m, 2H, CH₂), 4.74–4.81 (m, 2H, CH₂), 5.06–5.17 (m, 2H, CH₂), 5.73 (br, 1H, NH), 6.23–6.28 (m, 1H, = CH), 6.64–6.67 (m, 1H, = CH), 7.13 (d, J = 7.02 Hz, 1H, NH), 7.30–7.39 (m, 10H, ArCH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.06 (+ve, CH₃), 24.85 (−ve, CH_2Pro), 27.73 (−ve, CH_2Pro), 43.46 (−ve, CH_2Gly), 46.39 (−ve,CH_2Pro), 48.42 (+ve, CH_Ala), 60.12 (+ve, CH_Pro), 65.94 (−ve, CH₂), 66.96 (−ve, CH₂), 122.52 (+ve, ArCH), 126.67 (+ve, ArCH), 128.05 (+ve, ArCH), 128.15 (+ve, ArCH), 128.20 (+ve, ArCH), 128.53 (+ve, ArCH), 128.64 (+ve, ArCH), 134.68 (+ve, CH), 136.05 (ArC), 136.37 (ArC), 156.28 (C=O), 168.11 (C=O), 170.48 (C=O), 172.58 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₇H₃₁O₆N₃ ([M + Na]⁺) 516.2105, found 516.2094.

4.2.27. Cbz-Gly-Pro-Ala-O-Geranyl (26)

Compound 26 was synthesized according to the general procedure F as a brown thick oil, yield 84%; [α]²⁵_D −72.5° (0.4, CHCl₃); IR (ATR): 3309, 3063, 2931, 2363, 2117, 1997, 1647, 1535, 1446, 1326, 1252, 1051, 745, 700, 574 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.38 (d, J = 7.20 Hz, 3H, CH₃), 1.59 (s, 3H, CH₃), 1.68 (d, J = 8.95 Hz, 6H, 2 x CH_3Ger), 1.91–2.15 (m, 7H, 3H of CH_2Pro + 2 × CH_2Ger), 2.30–2.32 (m, 1H, CH_2Pro), 3.38–3.43 (m, 1H, CH_2Pro), 3.53–3.57 (m, 1H, CH_2Pro), 3.95–4.08 (m, 2H, CH_Ala + CH_Pro), 4.46–4.53 (m, 2H, CH₂), 4.53–4.68 (m, 2H, CH₂), 5.05–5.08 (m, 1H, CH), 5.10–5.12 (m, 2H, CH₂), 5.30–5.33 (m, 1H, CH), 5.71 (br, 1H, NH), 7.03–7.04 (m, 1H, NH), 7.31–7.36 (m, 5H, ArCH); DEPT-135 NMR (125 MHz, normal): δ 16.51 (+ve, CH₃), 17.72 (+ve, CH₃), 18.16 (+ve, CH₃), 24.86 (−ve, CH_2Pro), 25.70 (+ve, CH₃), 26.25 (−ve, CH₂), 27.78 (−ve, CH₂), 39.50 (−ve, CH₂), 43.46 (−ve, CH₂), 46.37 (−ve, CH₂), 48.40 (+ve, CH), 60.15 (+ve, CH), 62.29 (−ve, CH₂), 66.95 (−ve, CH₂), 117.76 (+ve, CH), 123.66 (+ve, CH), 128.04 (+ve, ArCH), 128.41 (+ve, ArCH), 128.52 (+ve, ArCH), 131.90 (ArC), 136.37 (ArC), 142.97 (ArC), 156.25 (C=O), 168.01 (C=O), 170.35 (C=O), 172.79 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₈H₃₉O₆N₃ ([M + H]⁺) 536.2731, found 536.2697.

4.2.28. Cbz-Gly-Pro-Ala-O-Crotyl (27)

Compound 27 was synthesized according to the general procedure F as a white solid, yield 81%, mp 103 °C; [α]²⁵_D −87.5° (0.4, CHCl₃); IR (ATR): 3302, 3063, 2987, 2363, 2117, 1997, 1722, 1446, 1654, 1535, 1259, 1155,, 1058, 976, 834, 611 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.29 (t, J = 7.09 Hz, 3H, CH₃), 1.43 (d, J = 7.22 Hz, 3H, CH_3Cro), 1.88–1.95 (m, 1H, CH_2Pro), 1.99–2.04 (m, 1H, CH_2Pro), 2.09–2.17 (m, 1H, CH_2Pro), 2.34–2.38 (m, 1H, CH_2Pro), 2.43–2.49 (m, 1H, CH_2Pro), 3.40 (q, J = 9.66 Hz, 1H, CH_2Pro), 3.54–3.58 (m, 1H, CH_2Pro), 3.97–4.07 (m, 2H, CH_Ala+Pro), 4.20 (q, J = 7.08 Hz, 2H, CH₂), 4.51–4.50 (m, 2H, CH₂), 4.78–4.80 (m, 2H, CH₂), 5.09–5.15 (m, 2H, CH₂), 5.72 (br, 1H, NH), 6.04 (d, J = 15.78 Hz, 1H, = CH), 6.92 (dt, J = 15.40 Hz, J = 4.60 Hz, 1H, = CH), 7.17 (d, J = 7.00 Hz, 1H, NH), 7.03–7.36 (m, 5H, ArH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 14.21 (+ve, CH_Cro), 17.94 (+ve, CH_3Ala), 24.88 (−ve, CH_2Pro), 27.49 (−ve, CH_2Pro), 43.46 (−ve, CH_2Gly), 46.43 (−ve, CH_2Pro), 48.35 (+ve, CH_Ala), 60.08 (+ve, CH_Pro), 60.69 (−ve, CH₂), 63.34 (−ve, OCH₂), 67.00 (−ve, CH₂), 122.58 (+ve, CH), 128.06 (+ve, ArCH), 128.17 (+ve, ArCH), 128.53 (+ve, ArCH), 136.34 (ArC), 140.40 (+ve, CH), 165.75 (C=O), 168.45 (C=O), 168.30 (C=O), 170.46 (C=O), 172.08 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₂₄H₃₁O₈N₃ ([M + H]⁺) 512.2003, found 512.2002.

4.2.29. Boc-Gly-OH (28)

Compound 28 was synthesized according to the general procedure A as a colorless liquid; yield 90%.

4.2.30. Boc-Gly-Pro-Ala-OMe (29)

Compound 29 was synthesized according to the general procedure C as a white solid; yield 40%, mp 155 °C; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.40 (d, J = 6.72 Hz, 3H, CH_3Ala), 1.45 (s, 9H, CH_3Boc), 1.88–1.95 (m, 1H, CH_2Pro), 2.12–2.14 (m, 1H, CH_2Pro), 2.33–2.37 (m, 1H, CH_2Pro), 3.39–3.41 (m, 1H, CH_2Pro), 3.52–3.56 (m, 1H, CH_2Pro), 3.74 (s, 3H, OCH₃), 3.89–4.01 (m, 2H, CH₂), 4.47–4.50 (m, 1H, CH_2Pro), 4.55–4.57 (m, 1H, CH), 5.42–5.44 (m, 1H, CH), 6.74 (d, J = 7.24 Hz, 1H, NH), 7.15 (d, J = 6.71 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 18.04 (+ve, CH_3Ala), 24.89 (−ve, CH_2Pro), 27.53 (−ve, CH_2Pro), 28.34 (+ve, CH_3Boc), 43.11 (−ve, CH_2Gly), 46.35 (−ve, CH_2Pro), 48.27 (+ve, CH_Ala), 52.46 (+ve, OCH₃), 60.06 (+ve, CH_Pro), 79.82 (+ve, C), 155.83 (C=O), 168.64 (C=O), 170.46 (C=O), 173.27 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₆H₂₇O₆N₃ ([M + Na]⁺) 380.1792, found 380.1792.

4.2.31. NH₂-Gly-Pro-Ala-OMe (30)

Compound 30 was synthesized according to the general procedure E as a light-yellow-colored liquid, yield 90%; [α]²⁵_D −132.5° (0.4, MeOH); IR (ATR): 3265, 3071, 2959, 2363, 2102, 1654, 1535, 1461, 1358, 1192, 984, 924, 834, 775, 514 cm^–1; ¹H NMR (500 MHz, CDCl₃, 25 °C, TMS): δ 1.29 (d, J = 7.27 Hz, 3H, CH_3Ala), 1.90–1.91 (m, 3H, CH_2Pro), 2.04–2.10 (m, 1H, CH_2Pro), 3.53–3.54 (m, 2H, CH_2Pro), 3.62 (s, 3H, OCH₃), 3.78–3.83 (m, 2H, CH₂), 4.20–4.26 (m, 1H, CH_Pro), 4.39 (dd, J = 8.71 Hz, 2.60 Hz, 1H, CH), 8.31 (br, NH₂), 8.47 (d, J = 7.33 Hz, 1H, NH); ¹³C NMR (125 MHz, normal/DEPT-135): δ 17.29 (+ve, CH_3Ala), 24.44 (−ve, CH_2Pro), 29.73 (−ve, CH_2Pro), 40.30 (−ve, CH_2Gly), 46.27 (−ve, CH_2Pro), 47.96 (+ve, CH_Ala), 52.36 (+ve, OCH₃), 59.55 (+ve, CH_Pro), 164.83 (C=O), 171.58 (C=O), 173.40 (C=O); HRMS (microTOF-QII, MS, ESI): calcd for C₁₁H₁₉O₄N₃ ([M + H]⁺) 258.1448, found 258.1457.

4.2.32. 2,2′-Bis(prop-2-yn-1-yloxy)-1,1′-binaphthalene (31)

Compound 31 was synthesized according to the general procedure G as a thick liquid; yield 95%; HRMS (microTOF-QII, MS, ESI): calcd for C₂₆H₁₈O₂ [M + Na]⁺ 385.4097, found 385.4070.

4.2.33. (3-Azidoallyl)benzene (32)

Compound 32 was synthesized according to the general procedure H as a yellowish-brown thick liquid; yield 80%; IR (KBr): 3459, 2862, 3028, 2937, 2094, 1886, 1736, 1669, 1490, 1386, 1237, 1088, 989, 879, 745, 693, 559 cm^–1.

4.2.34. 2,2′-Bis((1-((E)-3-phenylprop-1-en-1-yl)-1H-1,2,3-triazol-5-yl)methoxy)-1,1′-binaphthalene (33)

Compound 33 was synthesized according to the general procedure I as a yellowish-brown thick liquid, yield 95%; IR (KBr): 2922, 1595.2, 1326, 1215, 1043, 805, 752 cm^–1. ¹H NMR (500 MHz, CDCl₃) δ: 1.23–1.25 (s, 1H, CH), 2.02–2.06 (d, J = 16 Hz, 1H, CH), 4.80–4.81 (s, 4H, CH₂), 5.04–5.07 (m, 2H, CH₂), 5.17–5.19 (m, 2H, CH₂), 6.04–6.10 (m, 2H, ArH), 6.45 (s, 1H, ArH), 6.49 (s, 2H, ArH), 7.12 (s, 4H, ArH), 7.19–7.22 (m, 1H, ArH), 7.28–7.36 (m, 8H, ArH), 7.42–7.44 (d, J = 10 Hz, 2H, ArH), 7.68–7.69 (d, J = 5 Hz, 2H, ArH), 7.73–7.75 (d, J = 10 Hz, 2H, ArH); ¹³C NMR (125 MHz, normal/DEPT-135) δ: 14.32 (+ve, CH), 20.81 (+ve, CH), 21.07 (+ve, CH), 48.01 (−ve, CH₂), 52.15 (−ve, CH₂), 64.00 (−ve, CH₂), 64.08 (−ve, CH₂), 115.8 (+ve, ArCH), 115.99 (+ve, ArCH), 120.67 (+ve, ArC), 121.53 (+ve, ArCH), 122.19 (+ve, ArCH), 123.92 (+ve, ArCH), 125.35 (+ve, ArCH), 126.45 (+ve, ArCH), 126.80 (+ve, ArCH), 127.97 (+ve, ArCH), 128.63 (+ve, ArCH), 128.67 (+ve, ArC), 128.68 (+ve, ArCH), 128.80 (+ve, ArC), 128.81 (+ve, ArCH), 129.50 (+ve, ArCH), 129.51 (+ve, ArC), 133.90 (+ve, ArC), 134.06 (+ve, ArC), 135.34 (+ve, ArC). HRMS (microTOF-QII, MS, ESI): calcd for C₄₄H₃₆O₂N₆ ([M + H]⁺) 681.2772, found 681.2766.

4.3. AChE Inhibition Assay

The in vitro AChE activity of synthesized compounds was evaluated using the Ellman assay.³¹ Donepezil was used as a reference compound. Human AChE, acetylthiocholine iodide (ATChI), and 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich. Stock solutions of 10^–3 M concentration of all of the compounds were made in DMSO, and further dilutions to obtain the concentration range of 10^–5–10^–8 M were made in PBS buffer. AChE was dissolved in PBS (NaH₂PO₄/Na₂HPO₄) buffer to obtain a solution of 2.5 units/mL at pH 7.2. DTNB and ATChI solutions of 0.3 mM and 10 mM concentrations, respectively, were prepared in PBS buffer. 20 μL of AChE and 140 μL of DTNB (0.3 mM) solutions were added to the 96-well plate with or without test compounds (20 μL). The blank well contains all of the components as in the test wells except for the enzyme. The resulting reaction mixture was incubated at 25 °C for 20 min, followed by the addition of 20 μL of ATChI solution (10 mM). The absorbance of the reaction mixture at 412 nm was recorded on a microplate reader (Synergy BioTek 96-well microplate reader) at 1 min interval for a total of 10 min. All experiments were run in triplicate. The %age inhibition of the enzyme by the respective test compounds was determined using the equation 100 – (T/C) × 100, where T and C represent enzyme activity with or without the test compound, respectively. IC₅₀ values were obtained graphically and are reported as mean ± SEM.

4.4. BACE-1 inhibition assay

The BACE-1 fluorescence resonance energy transfer (FRET) assay kit was purchased from Sigma-Aldrich, and the assay was performed according to the supplier’s protocol. Stock solutions of the substrate (500 μM) and the test compounds (10 mM) were prepared using DMSO and kept at 20 °C. Further dilutions of the test compounds were done in a fluorescent assay buffer (FAB) supplied in the kit. BACE-1 enzyme solution (0.3 unit/L) and 50 μM substrate solution were produced by diluting with the fluorescent assay buffer just before starting the experiment. With excitation at 320 nm and emission at 405 nm, the fluorometer was set to the well plate reader mode. In the assay, a 96-well black polystyrene microplate was filled with 10 μL of the test compound, 20 μL of substrate, and 68 μL of FAB. The time zero reading of fluorescence was measured immediately after the addition of 2 μL of BACE-1. Then, the plate was incubated at 37 °C and the fluorescence was measured after 2 h. Triplicates of each concentration were tested. In the control wells, all chemical reagents except for BACE-1 were added. The background signal was measured using the fluorescence readings of the control wells and subtracted from all of the wells containing test compounds. Also, the fluorescence readings of the assay solutions without inhibitors were taken. The following expression was used to compute the percentage of inhibition caused by the presence of the test compound: 100- (Fi/Fo x 100), where Fi and Fo are the fluorescence intensities obtained in the presence and absence of inhibitors, respectively.

Calbiochem IV (CAS no. 797035-11-1) was taken as the reference drug with IC₅₀ of 18 nM (literature value).

4.5. Inhibition of Aβ_1–42 Aggregation

Inhibition of Aβ_1–42 aggregation by the synthesized compounds was evaluated using a Thioflavin-T (ThT) fluorometric assay.⁴¹ Recombinant human HFIP-pretreated Aβ_1–42 peptide (7.5 μM) was prepared in 150 mM HEPES buffer of pH 7.4. Donepezil was used as the standard drug. Stock solutions of concentration 100 μM of all of the test compounds along with donepezil were prepared in HEPES buffer. 20 μL of HFIP (7.5 μM) was mixed with 10 μL of the test compounds and donepezil and subsequently added to the corresponding wells of black-walled 96-well plates in quadruplicate. Then, 70 μL of ThT solution of 14.3 μM concentration was added to each well, making a final volume of 100 μL in each well. The kinetic experiment with an excitation wavelength of 440 nm and emission wavelength of 490 nm for 48 h was run on a 96-well microplate reader (Synergy Hybrid reader, BioTek Instruments). The ThT fluorescence through the bottom of the plate was measured every 3 min with a medium continuously shaking between the measurements. The fluorescence intensities at the plateau were averaged for all of the test compounds, and the average fluorescence at t = 0 h was subtracted. The percentage inhibition of the Aβ_1–42 self-induced aggregation was calculated using the formula

where F_i is the increase in fluorescence of Aβ_1–42 treated with the test compounds and F₀ is the increase in the fluorescence of Aβ_1–42 alone.

4.6. PAMPA

The used 96-well precoated PAMPA Plate System Gentest was obtained from Corning (Catalog number 353015). Caffeine and norfloxacin were used as the reference compounds. Solutions of all of the tested compounds (200 μM) were prepared in PBS buffer (pH 7.4). 300 μL of the test compound solution was added to the donor wells, whereas 200 μL of buffer was added to all wells of the acceptor plate. All of the compounds were tested in triplicate. The plate was incubated for 5 h at room temperature. Then, 150 μL was aspirated from each well, and the concentrations of the compounds in the acceptor and donor wells were calculated with LC-MS. The permeability coefficients (Pe) were calculated according to the formulas given in the protocol. According to the PAMPA plate’s manufacturer, compounds with Pe values higher than 1.5 × 10^–6 cm/s possess good human oral absorption capacity.

4.7. Molecular Docking

Molecular docking studies were performed by using the Schrödinger software package (Schrödinger Release 2015-4: Maestro, version 10.0, Schrödinger, LLC). The 3D crystal coordinates of AChE, BACE-1, and Aβ were retrieved from the protein data bank (www.rcsb.org) with pdb IDs 1EVE, 4B05, and 1IYT, respectively. The proteins were preprocessed, polar hydrogens were added, and water molecules were removed. Taking into account the drug solubility and permeability, the heteroatoms were ionized at biological pH. To reduce steric clashes by amino acids, hydrogen bonds were optimized. Using the Ligprep tool of Schrödinger, ligands were prepared, which adapt a 3D structure. Prior to docking, a cubic grid dimension with various dimensions with the grid points along the x, y, and z axes was generated with a van der Waals radius of 1. The ligands were then docked into the generated grids, and the interactions of the prepared ligand with the receptor were calculated using the XP ligand docking in glide. Interactions between ligands and enzymes were analyzed, and hydrogen atoms that are nonpolar were merged to carbon atoms. The docking procedure was validated by docking the cocrystallized ligands of AChE and BACE-1 in the respective enzymes, and RMSD was calculated.

4.8. Animal Studies

Swiss albino mice (25–30 g) were purchased from the Indian Institute of Integrative Medicine, Jammu, India. During the entire studies, animals had free access to water and feed. All of the animal experiments were approved by the Institutional Animal Ethics Committee constituted under CPCSEA with reference no. 226/CPCSEA/2019/47.

4.9. Experiment Design for Behavioral Analysis

The Morris water maze (MWM) test and the passive avoidance task (PAT) were used for the behavioral studies of mice in order to evaluate the effect of the new compounds. In this study, mice were placed into eight groups, each having five animals. Group 1 was taken as the control, and groups 2–8 were given scopolamine, donepezil, compound 15, compound 18, compound 25, compound 26, and compound 30, respectively. The animals were trained for 4 days in the MWM test. On the fifth day, the basal readings were taken. The untrained animals were excluded from the studies. Various interventions (0.2 mL of 10 mg/kg conc) were given for a total of 9 days, starting from day 6. Scopolamine (2 mg/kg, i.p.) treatment was given half an hour prior to the various interventions on the 12th, 13th, and 14th days of the experiment. The behavioral observation was taken on day 11 (before scopolamine treatment) and day 14 (after scopolamine treatment). On the 14th day, 1 h after behavioral analysis, the animals were sacrificed and the brain tissues were harvested for biochemical tests.⁴² The overall experimental design is illustrated in Figure 8.

Figure 8.

Overall experimental design for the memory test of the animals.

4.10. Morris Water Maze (MWM) Test

Morris water maze is a circular tank having 90 cm diameter and 50 cm height. The tank is divided into four quadrants with Any-Maze software. One out of the four quadrants has a circular platform of 9 cm diameter. The tank was filled with water (maintained at 25 °C) in such a manner that the water level remains 2 cm above the platform. To hide the platform, the water in the tank was made opaque with nontoxic colors. The camera connected to Any-Maze software was mounted above the tank. The track of animal movement was recorded with software (ANY-maze 5.7, Ugo Basile, Italy). A 10 s of stay on the platform was regarded as a correct entry. A latency to reach the platform was taken as a measure of animal memory.⁴³

4.11. Passive Avoidance Task (PAT)

The passive avoidance task apparatus is a square box equipped with small electrical rods on the floor having a square-shaped shock-free area. The electrical rods were kept on a low-intensity current, and the animal was placed in one corner of the apparatus. The animal was placed on the grid floor, and a low-intensity current was supplied to the grid. The latency to reach a shock-free area (s) was recorded and taken as a measure of animal memory. A correct entry to the shock-free area was registered if all of the paws of mice were on the shock-free area.⁴⁴

4.12. Plasma Stability Assay

A stock solution of compound 25 (5 mM) was prepared in DMSO. Ultrapure water was used to prepare PBS buffer (pH 7.4; 0.01M, 0.8 g of NaCl, 0.03 g of KCl, 0.14 g of Na₂HPO₄, 0.024 KH₂PO₄). A solution of compound 25 in PBS (50 μM) was used as the positive control. Plasma was extracted by centrifuging the blood sample at 3000 rpm for 15 min at 4 °C. Then, the supernatant was collected using a micropipette and stored at −20 °C. Before its use, plasma was centrifuged at 2000 rpm for 10 min to remove fibrous precipitates resulting from freezing, and the supernatant was retained. Plasma (pH 7–8) was treated with 25 to produce the solution of final concentration 50 μM. After incubation of the sample at 37 °C for 1 and 12 h, 400 μL of acetonitrile was added to quench the reaction. Then, the sample was centrifuged at 10,000 rpm at room temperature for 15 min and the supernatant was separated. To quantify the concentration of the compound in plasma, 100 μL of treated supernatant and buffer solutions were injected into the spectrometer using an autosampler. The mobile phase gradient comprised 100% acetonitrile. The spectra were recorded in the positive ion mode. The percentage of analyte lysed by the plasma of each solution was calculated from intensities of peaks observed from the HRMS spectra at the same interval of time using eq 1, where I₀ and I represent the intensities of the compound in the presence of buffer and plasma (treated supernatant). Remarkably, a slight change in the concentration of 25 (∼10%) was observed after a long time of incubation (12 h). It is demonstrated that plasma had no remarkable effect on the half-life time of 25, and further, it can be prioritized for in vivo studies.

4.13. Lipinski Parameters

Lipinski parameters and ADME properties of the designed compounds were checked using online softwares Molinspiration⁴⁵ and PreADMET,⁴⁶ respectively.

Glossary

Abbreviations

CNS

central nervous system

AD

Alzheimer’s disease

BACE-1

β-site APP cleaving enzyme 1

AChE

acetylcholine esterase

PNS

peripheral nervous system

Boc

t-butyloxycarbonyl

TEA

triethylamine

ECF

ethyl chloroformate

ACN

acetonitrile

Cbz

benzyloxy carbonyl

THF

tetrahydrofuran

DCM

dichloromethane

DMF

dimethylformamide

Aβ

amyloid β

PAMPA

β-site APP cleaving enzyme 1

MWM

Morris water maze

PAT

passive avoidance task

ADME

absorption distribution metabolism excretion

IC₅₀

concentration for 50% inhibition

TPSA

total polar surface area

log P

partition coefficient

TLC

thin-layer chromatography

SEM

standard error mean

i.p.

intraperitoneally

DMSO

dimethyl sulfoxide

TFA

trifluoroacetic acid

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c09069.

• ¹H and ¹³C NMR spectra, mass spectra, IR spectra and molecular docking data (PDF)

• Three-dimensional model of target–ligand (compounds docked in BACE-1) (ZIP)

• Three-dimensional model of target–ligand (compounds docked in AChE) (ZIP)

• PDB coordinates for computational models (ZIP)

• Molecular formula strings (CSV)

Author Present Address

^§ Medicinal Chemistry, Virginia Commonwealth University, 300 West Franklin Street, Richmond, Virginia 23220, United States

The authors declare no competing financial interest.