Design, Synthesis, and Evaluation of Novel Phenolic Acid/Dipeptide/Borneol Hybrids as Potent Angiotensin Converting Enzyme (ACE) Inhibitors with Anti-hypertension Activity

Under the guidance of combination of traditional Chinese medicine chemistry (CTCMC), this study describes the preparation of a phenolic acid/dipeptide/borneol hybrid consisting of phenolic acid and a bornyl moiety connected to the dipeptide N-terminal and C-terminal respectively. It also evaluates their angiotensin converting enzyme (ACE) inhibitory and synergistic antihypertensive activities. Briefly, a series of novel phenolic acid/dipeptide/borneol hybrids were prepared and investigated for their ability to inhibit ACE. The influence of the phenolic acid and bornyl moiety on subsite selectivity is also demonstrated. Among all the new compounds, two compounds—7a and 7g—reveal good inhibition potency in in vitro ACE-inhibitory tests. Interestingly, favorable binding results in molecular docking studies also supported the in vitro results. Additionally, the bioassay showed that oral administration of the two compounds displayed high and long-lasting antihypertensive activity both in acute antihypertensive tests and in therapeutic antihypertensive tests by non-invasive blood pressure measurements in spontaneously hypertensive rats.


Introduction
Hypertension is a metabolic cardiovascular syndrome that affects nearly one billion people around the world [1,2]. Clinically, it is the major risk factor for various heart diseases, such as arteriosclerosis, coronary heart disease, stroke, peripheral arterial disease, and heart failure [3,4]. Nowadays, the angiotensin converting enzyme (ACE) inhibitors (e.g., Capoten (captopril), Vasotec (enalapril), Prinivil, or Zestril (lisinopril), etc.) have been considered as first-line drugs for the treatment of hypertension and myocardial infarction [5,6]. However, it was found that any single currently used ACE inhibitor cannot completely prevent and cure hypertension. Clinically, they are usually used in conjunction with one or two other therapeutic agents including beta blockers, long-acting nitrate, and calcium channel blockers for a better therapeutic effect (e.g., Enalapril/hydrochlorothiazide, Enalapril/Valsartan, and Enalapril/nifedipine, etc.). Moreover, ACE inhibitors are also found to have some side effects, such as Enalapril/Valsartan, and Enalapril/nifedipine, etc.). Moreover, ACE inhibitors are also found to have some side effects, such as cough, elevated blood potassium levels, low blood pressure, and kidney failure [7,8]. Therefore, the trend has been toward developing safer and multi-functional ACE inhibitors.
Many pioneering work in the mid-1970s to mid-1990s revealed that the N-substituted derivative of proline is an extremely potent ACE inhibitor [9][10][11][12]. Recently, during our study on compound Salvia recipe (including three Chinese herbal medicines: Salvia miltiorrhiza Bge. (Danshen), Panax pseudoginseng Wall. (Sanqi), and Borneol (Bingpian, from Cinnamomum camphora (L.) Presl)) for searching new antihypertensive drugs, we noticed that the phenolic acids extract from Danshen plays a pivotal role in regulating blood pressure, protecting against oxidative damage diseases, slightly slowing the heart rate, and relaxing and widening blood vessels [13], while borneol may help other medicinal molecules to penetrate cell membrane easily [14,15]. In fact, the molecular dimension of some phenolic acids, especial those structures varying from C6-C1 to C6-C3, are more suitable for ACE S1 subsite [16] and the hydroxyls on these phenolic acids may serve as good hydrogen donors to chelate with Zn 2+ in the active site of ACE [17]. More recently, some phenolic acids, such as ferulic acid, gallic acid, chlorogenic acid, and caffeic acid, have been demonstrated to be a new class of selective inhibitors of ACE [18,19]. So, in this study, based on the effectiveness of compound Salvia recipe in the prevention and treatment of angiocardiopathy and the ideas of CTCMC, we tried to add these effective phenolic acid and bornyl moieties simultaneously to the N-terminal and C-terminal of dipeptides (Val or Leu-Pro, the core structure of some lead ACE inhibitors [20][21][22]) (see Figure 1) by simply using amide and ester bonds, respectively. We envision that this molecular construction strategy may result in producing more novel lead compounds with multi-functions, not only targeting ACE, but also other targets relating to synergistic hypotensive effects, which can overcome some side effects of current ACE inhibitors.

Synthesis and Preparation of 7a-7h
Commercially available L-proline 1 was initially converted to N-Boc-Proline 2 in the presence of di-tert-butyl dicarbonate in good yield and then followed by bornyl ester formation under Steglich esterification conditions to afford N-Boc-Pro-bornyl ester 3. This was subjected to trifluoracetic acid(TFA)/dichloromethane(DCM) conditions to afford compound 4, which was coupled with N-Boc-Val and N-Boc-Leu respectively under the 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride(EDCI)/1-hydroxybenzotriazole (HOBT) system to furnish the corresponding N-Bocdipeptide ester 5a and 5b. Removal of the Boc group of 5a and 5b was accomplished with the above mentioned TFA/DCM conditions to give 6a and 6b in near-quantitative yields. Finally, the target compounds 7a-7h were achieved by general coupling reaction of 5a and 5b with corresponding phenolic acids under the benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(Pybop)/N,Ndiisopropyl-ethylamine (DIPEA) system. The synthesis of the new ACE inhibitors 7a-7h is shown in Scheme 1.

Synthesis and Preparation of 7a-7h
Commercially available L-proline 1 was initially converted to N-Boc-Proline 2 in the presence of di-tert-butyl dicarbonate in good yield and then followed by bornyl ester formation under Steglich esterification conditions to afford N-Boc-Pro-bornyl ester 3. This was subjected to trifluoracetic acid(TFA)/dichloromethane(DCM) conditions to afford compound 4, which was coupled with N-Boc-Val and N-Boc-Leu respectively under the 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride(EDCI)/1-hydroxybenzotriazole (HOBT) system to furnish the corresponding N-Boc-dipeptide ester 5a and 5b. Removal of the Boc group of 5a and 5b was accomplished with the above mentioned TFA/DCM conditions to give 6a and 6b in near-quantitative yields. Finally, the target compounds 7a-7h were achieved by general coupling reaction of 5a and 5b with corresponding phenolic acids under the benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(Pybop)/N,N-diisopropyl-ethylamine (DIPEA) system. The synthesis of the new ACE inhibitors 7a-7h is shown in Scheme 1.

In Vitro ACE-Inhibitory Activity
The in vitro ACE-inhibitory activity of the newly synthesized compounds was evaluated using ACE inhibition assay (Sigma-aldrich, St. Louis, MO, USA, No. A-6778). The experiments were carried out for all the designed compounds (7a-7h), for ACE-inhibitory activity at five different concentrations (0.01-10 µM). Lisinopril, a well-known ACE inhibitor drug, was used as standard for comparison with the inhibitory activity of synthesized compounds. The ACE-inhibitory activity can be calculated by the following equation: ACE-inhibitory activity (inhibition rate %) = [(Ablank1 − Asample)/(Ablank1 − Ablank2)] × 100, where blank 1: Positive control (without ACE inhibition) and blank 2: Reagent blank. Then we measured and calculated the IC50 values of the designed compounds (7a-7h).

Molecular Docking Study
To gain insight into the interaction of ACE with the most potent compound, it was docked on to tACE (PDB code: 1O86) using AutoDock Vina software (v4.2) (The Scripps Research Institute, San Diego, CA, USA). Structures of the ligand were generated using ChemBioDraw Ultra (v13.0) (Cambridgesoft, Billerica, MA, USA).

Antihypertensive Tests In Vivo
All the experiments were approved by the Institutional Animal Ethics Committee of Northwest University. Experiments were carried out on spontaneously hypertension rats (SHRs) that were 9-10 weeks old and weighed 200-220 g. SHRs treated with normal saline were used as controls. Lisinopril was used as a standard drug. Blood pressure (BP) was recorded automatically every day by the indirect tail-cuff method using an electrosphygmomanometer (BP-2010A Storage Pressure Meter). In acute antihypertensive tests in vivo, the most potent and active compounds obtained in in vitro tests were measured using spontaneously hypertensive rats. The BP were measured before and 0.5, 1, 2, 3, 4, 5 and 6 h after a single dose of intragastrical administration of active compounds. In therapeutic antihypertensive tests, the more potent and active compound and lisinopril were treated orally for 7 consecutive days, and the BP was measured every day between drug delivery day and 14 days after the drug treatment using an intelligent non-invasive blood pressure remote monitoring instrument.

In Vitro ACE-Inhibitory Activity
The in vitro ACE-inhibitory activity of the newly synthesized compounds was evaluated using ACE inhibition assay (Sigma-aldrich, St. Louis, MO, USA, No. A-6778). The experiments were carried out for all the designed compounds (7a-7h), for ACE-inhibitory activity at five different concentrations (0.01-10 µM). Lisinopril, a well-known ACE inhibitor drug, was used as standard for comparison with the inhibitory activity of synthesized compounds. The ACE-inhibitory activity can be calculated by the following equation: ACE-inhibitory activity (inhibition rate %) = [(A blank1 − A sample )/(A blank1 − A blank2 )] × 100, where blank 1: Positive control (without ACE inhibition) and blank 2: Reagent blank. Then we measured and calculated the IC 50 values of the designed compounds (7a-7h).

Molecular Docking Study
To gain insight into the interaction of ACE with the most potent compound, it was docked on to tACE (PDB code: 1O86) using AutoDock Vina software (v4.2) (The Scripps Research Institute, San Diego, CA, USA). Structures of the ligand were generated using ChemBioDraw Ultra (v13.0) (Cambridgesoft, Billerica, MA, USA).

Antihypertensive Tests In Vivo
All the experiments were approved by the Institutional Animal Ethics Committee of Northwest University. Experiments were carried out on spontaneously hypertension rats (SHRs) that were 9-10 weeks old and weighed 200-220 g. SHRs treated with normal saline were used as controls. Lisinopril was used as a standard drug. Blood pressure (BP) was recorded automatically every day by the indirect tail-cuff method using an electrosphygmomanometer (BP-2010A Storage Pressure Meter). In acute antihypertensive tests in vivo, the most potent and active compounds obtained in in vitro tests were measured using spontaneously hypertensive rats. The BP were measured before and 0.5, 1, 2, 3, 4, 5 and 6 h after a single dose of intragastrical administration of active compounds. In therapeutic antihypertensive tests, the more potent and active compound and lisinopril were treated orally for 7 consecutive days, and the BP was measured every day between drug delivery day and 14 days after the drug treatment using an intelligent non-invasive blood pressure remote monitoring instrument.

Chemistry
The synthesis of target compounds 7a-7h was achieved following the procedures outlined in Scheme 1. The chemical structure, synthetic conditions and yield of 7a-7h are shown in Scheme 2.

Chemistry
The synthesis of target compounds 7a-7h was achieved following the procedures outlined in Scheme 1. The chemical structure, synthetic conditions and yield of 7a-7h are shown in Scheme 2.

ACE-Inhibitory Activity In Vitro
The preliminary biological investigations were conducted in vitro using the ACE-inhibition assay. The experiments carried out for all the designed compounds (7a-7h), for ACE-inhibitory activity at five different concentrations (0.01-10 µM), revealed that these newly synthesized compounds inhibited ACE in a dose-dependent manner (Figure 2). The evaluation results of all eight new compounds for their ACE-inhibitory activity are presented in Table 1. Unfortunately, all of these new compounds showed a comparatively lower ACE-inhibitory activity. Relatively, evaluation of IC 50 data against ACE for these compounds revealed that 7a and 7g are the best candidates and more encouraging compounds with the lowest IC 50 values of 6.76 and 6.88 µM, respectively. In contrast, the size of the amino acid side chain had a great influence on the activity of these compounds. That is, the activity of these compounds gradually declined when the size of the amino acid side chain increased. Besides, the influence of phenolic acids on ACE-inhibitory activity was more obvious following the order: protocatechuic acid > ferulic acid > gallic acid > 3,4-dihydroxybenzene acid. That is, substituents in positions 3 and 4 in the benzene ring are beneficial to the increase of ACE-inhibitory activity. At the same time, the shorter the amino side chain in phenolic acids, the better the ACE-inhibitory activity.

ACE-Inhibitory Activity In Vitro
The preliminary biological investigations were conducted in vitro using the ACE-inhibition assay. The experiments carried out for all the designed compounds (7a-7h), for ACE-inhibitory activity at five different concentrations (0.01-10 µM), revealed that these newly synthesized compounds inhibited ACE in a dose-dependent manner ( Figure 2). The evaluation results of all eight new compounds for their ACE-inhibitory activity are presented in Table 1. Unfortunately, all of these new compounds showed a comparatively lower ACE-inhibitory activity. Relatively, evaluation of IC50 data against ACE for these compounds revealed that 7a and 7g are the best candidates and more encouraging compounds with the lowest IC50 values of 6.76 and 6.88 µM, respectively. In contrast, the size of the amino acid side chain had a great influence on the activity of these compounds. That is, the activity of these compounds gradually declined when the size of the amino acid side chain increased. Besides, the influence of phenolic acids on ACE-inhibitory activity was more obvious following the order: protocatechuic acid > ferulic acid > gallic acid > 3,4-dihydroxybenzene acid. That is, substituents in positions 3 and 4 in the benzene ring are beneficial to the increase of ACE-inhibitory activity. At the same time, the shorter the amino side chain in phenolic acids, the better the ACEinhibitory activity. activity. At the same time, the shorter the amino side chain in phenolic acids, the better the ACEinhibitory activity. 6.76 ± 0.96 7b activity. At the same time, the shorter the amino side chain in phenolic acids, the better the ACEinhibitory activity.

ACE Molecular Docking
Based on the inhibition results, we selected the most potent compound 7a, our best ACE inhibitor in this study, as a ligand example. To gain insight into the interaction of ACE with compound 7a, it was docked on to tACE (PDB code: 1O86) using AutoDock Vina software [23]. As shown in Figure 3A, 7a and ligand compound (Lisinopril) of tACE can nearly overlap in the binding model. That is, they have the same binding sites and have a similar combination model. The intermolecular interactions between tACE binding site residues and predicted poses for compound 7a are presented in Figure 3B. Results showed that the docking position of compound 7a showed a similar binding mode to that of lisinopril. The intermolecular hydrogen-bonding interactions of 7a were mainly seen with the Gln281, His 353, His 513, Tyr 522 and Tyr 523, which are partly similar to lisinopril.

Antihypertensive Tests In Vivo
In acute antihypertensive tests in vivo, the more potent and active compounds 7a and 7g were investigated further using spontaneously hypertensive rats (SHRs). Lisinopril was used as a positive

ACE Molecular Docking
Based on the inhibition results, we selected the most potent compound 7a, our best ACE inhibitor in this study, as a ligand example. To gain insight into the interaction of ACE with compound 7a, it was docked on to tACE (PDB code: 1O86) using AutoDock Vina software [23]. As shown in Figure 3A, 7a and ligand compound (Lisinopril) of tACE can nearly overlap in the binding model. That is, they have the same binding sites and have a similar combination model. The intermolecular interactions between tACE binding site residues and predicted poses for compound 7a are presented in Figure 3B. Results showed that the docking position of compound 7a showed a similar binding mode to that of lisinopril. The intermolecular hydrogen-bonding interactions of 7a were mainly seen with the Gln281, His 353, His 513, Tyr 522 and Tyr 523, which are partly similar to lisinopril.

ACE Molecular Docking
Based on the inhibition results, we selected the most potent compound 7a, our best ACE inhibitor in this study, as a ligand example. To gain insight into the interaction of ACE with compound 7a, it was docked on to tACE (PDB code: 1O86) using AutoDock Vina software [23]. As shown in Figure 3A, 7a and ligand compound (Lisinopril) of tACE can nearly overlap in the binding model. That is, they have the same binding sites and have a similar combination model. The intermolecular interactions between tACE binding site residues and predicted poses for compound 7a are presented in Figure 3B. Results showed that the docking position of compound 7a showed a similar binding mode to that of lisinopril. The intermolecular hydrogen-bonding interactions of 7a were mainly seen with the Gln281, His 353, His 513, Tyr 522 and Tyr 523, which are partly similar to lisinopril.

Antihypertensive Tests In Vivo
In acute antihypertensive tests in vivo, the more potent and active compounds 7a and 7g were investigated further using spontaneously hypertensive rats (SHRs). Lisinopril was used as a positive

Antihypertensive Tests In Vivo
In acute antihypertensive tests in vivo, the more potent and active compounds 7a and 7g were investigated further using spontaneously hypertensive rats (SHRs). Lisinopril was used as a positive control. The reduction in blood pressure (BP, systolic arterial pressure) of SHRs treated with compounds 7a and 7g at the same molar dose was slightly better than that of lisinopril under similar conditions. Additionally, the antihypertensive activity of 7a and 7g occurred evidently 0.5 h after oral treatment and lasted for four hours ( Figure 4A,B). We then assessed the effects of compounds 7a and 7g in therapeutic antihypertensive tests in SHRs. We found that BP in compound 7a-and 7g-treated (100 mg/kg, Po. 7days) rats was decreased remarkably on the second day after oral administration. Then, the BP maintained at a relatively stable level. After drug withdrawal, the BP on the eighth day remained at a lower level. However, the BP began to rebound starting from the ninth day, and it returned back to the level before administration on the fourth day after drug withdrawal. Thus, compounds 7a and 7g showed remarkable hypotensive activity in SHRs ( Figure 4C), and the maximum antihypertensive effect of the two compounds at a dose of 100 mg/kg daily was also approximately equivalent to that of lisinopril (20 mg/kg/day). It is worth noting that successive dosing has no effect on the body weight of the test rats in therapeutic antihypertensive tests. control. The reduction in blood pressure (BP, systolic arterial pressure) of SHRs treated with compounds 7a and 7g at the same molar dose was slightly better than that of lisinopril under similar conditions. Additionally, the antihypertensive activity of 7a and 7g occurred evidently 0.5 h after oral treatment and lasted for four hours ( Figure 4A,B). We then assessed the effects of compounds 7a and 7g in therapeutic antihypertensive tests in SHRs. We found that BP in compound 7a-and 7g-treated (100 mg/kg, Po. 7days) rats was decreased remarkably on the second day after oral administration. Then, the BP maintained at a relatively stable level. After drug withdrawal, the BP on the eighth day remained at a lower level. However, the BP began to rebound starting from the ninth day, and it returned back to the level before administration on the fourth day after drug withdrawal. Thus, compounds 7a and 7g showed remarkable hypotensive activity in SHRs ( Figure 4C), and the maximum antihypertensive effect of the two compounds at a dose of 100 mg/kg daily was also approximately equivalent to that of lisinopril (20 mg/kg/day). It is worth noting that successive dosing has no effect on the body weight of the test rats in therapeutic antihypertensive tests.  (A,B), the acute antihypertensive activity of compounds 7a and 7g in spontaneously hypertension rats (SHRs) for single dose administration (dosing time: 0 min); (C) the therapeutic antihypertensive activity of compounds 7a and 7g in SHRs for 7 days treatment (dosing time: 0 day; blood pressure (BP) measured time: 9:30-11:30 am). Data are presented as mean ± SEM, n = 6. The BP was determined with a BP-2010A Storage Pressure Meter. This system measures BP by recording the cuff pressure at which the interrupted blood flow returns to the tail. Before drug administration, training consisted of five sessions over 3 days. The SHRs were placed into a thermostatically controlled Lucite box of the Visitech system BP2010A, and heated on a heated pad maintained at 37 °C for 2 min before BP measurement started. The tail root of SHR was surrounded by an inflatable tubular sleeve, and a pulse electron detector was placed in the root of the tails. Then BP was measured after the computer provided a stable signal. The BP for the session was accepted as the average of five BP readings that were within 10 mm Hg. The average BP values were calculated by employing the ANOVA method.

Conclusions
In conclusion, we developed an efficient and shortcut method for drug discovery based on both the means of combinatorial chemistry and pharmaceutical design philosophy of traditional Chinese  (A,B), the acute antihypertensive activity of compounds 7a and 7g in spontaneously hypertension rats (SHRs) for single dose administration (dosing time: 0 min); (C) the therapeutic antihypertensive activity of compounds 7a and 7g in SHRs for 7 days treatment (dosing time: 0 day; blood pressure (BP) measured time: 9:30-11:30 am). Data are presented as mean ± SEM, n = 6. The BP was determined with a BP-2010A Storage Pressure Meter. This system measures BP by recording the cuff pressure at which the interrupted blood flow returns to the tail. Before drug administration, training consisted of five sessions over 3 days. The SHRs were placed into a thermostatically controlled Lucite box of the Visitech system BP2010A, and heated on a heated pad maintained at 37 • C for 2 min before BP measurement started. The tail root of SHR was surrounded by an inflatable tubular sleeve, and a pulse electron detector was placed in the root of the tails. Then BP was measured after the computer provided a stable signal. The BP for the session was accepted as the average of five BP readings that were within 10 mm Hg. The average BP values were calculated by employing the ANOVA method.

Conclusions
In conclusion, we developed an efficient and shortcut method for drug discovery based on both the means of combinatorial chemistry and pharmaceutical design philosophy of traditional Chinese medicine. We call this method combination of traditional Chinese medicine chemistry (CTCMC). Using this method, and inspired by the "Danshen-borneol" drug pair and some classical ACE inhibitors, we designed eight novel peptidomimetics (phenolic acid-Pro-borneol) compounds, which were successively synthesized and evaluated in vitro and in vivo as potential ACE inhibitors with less side effects. Of these, bornyl esters of 7a and 7g exhibited good in vivo ACE-inhibitory activity. These two compounds are most likely acting as prodrugs since both were found to have moderate activity in vitro as inhibitors of ACE. Based on these preliminary and promising results, more suitable modifications in the phenolic moieties and amino acid residue adjoining proline are under development. This class of novel molecules, combined with their detailed antihypertensive activities, will be useful for future drug development and shall be presented in the near future.