INTRODUCTION:

Hypertension is a common and often progressive disorder that poses a significant risk for cardiovascular and renal diseases. From a pathophysiological viewpoint, hypertensive disease involves changes in at least one of the hemodynamic variables (cardiac output, arterial stiffness, or peripheral resistance) that determine measurable blood pressure^{(1, 2)}.

The renin-angiotensin system (RAS) is a complex regulatory hormonal system with many identifiable actions ⁽³⁾, particularly in the control of hemodynamic stability by regulation of blood pressure, fluid volume, and sodium-potassium balance ^{(4, 5)}. Although renin is synthesized as a prohormone in the kidney, ⁽⁶⁾ it is also synthesized in many other tissues, including the brain, adrenal gland, ovary, and visceral adipose tissue, and possibly the heart and vascular tissue ^{(3, 7)}. Only renin-expressing cells in the kidney are capable of generating active renin from pro-renin ⁽⁸⁾.

In circulation, renin cleaves angiotensinogen (AGT) to produce angiotensin I (Ang I), which again has no biological action ⁽⁹⁾. Angiotensin I is hydrolyzed into a potent vasopressor peptide, angiotensin II (Ang II), by angiotensin-converting enzyme (ACE)⁽¹⁰⁾. ACE is a zinc-and chloride-dependent dipeptidyl carboxypeptidase I (DCP1), also known as kininase II, peptidyl-dipeptidase I, peptidase p, and carboxycathepsin ⁽¹¹⁾. ACE was first identified as a vital component of the renin-angiotensin and kallikrein-kinin systems (KKS) ⁽¹²⁾. It plays a significant role in blood pressure regulation, fluid and electrolyte balance, cardiovascular system development, and vascular remodeling. ACE also has a role in deactivation of the vasodepressor effect of peptide bradykinin⁽¹²⁾.

Captopril was the first orally active ACE inhibitor developed in 1975, mainly for the treatment of hypertension and congestive heart failure^{(13, 14)}. Captopril is also likely to cause an increase in plasma renin activity, due to a loss of feedback inhibition mediated by Ang II on the release of renin and stimulation of reflex mechanisms via baroreceptors. Decreased concentrations of aldosterone are found in the blood and urine, and as a result, small increases in serum potassium may occur along with sodium and fluid loss⁽¹⁵⁾. Captopril exhibits highly specific enzyme inhibition and receptor interactions. For example, captopril concentrations in the 40,000 to 70,000 fold range required to inhibit ACE are usually needed to inhibit some peptidases, such as carboxypeptidase A and B, trypsin, and chymotrypsin ^{(16, 17)}, and captopril has approximately 30,000-fold higher ACE affinity than Angiotensin-1 receptor (AT1R) ⁽¹⁸⁾.

In addition to the antihypertensive effects of captopril, findings have shown many other pharmacological applications, such as anti-inflammatory ⁽¹⁹⁾, antioxidant⁽²⁰⁾, and antiplatelet ⁽²¹⁾ with limited adverse effects as sore throat, dry cough, and short half-life to reduce patient compliance⁽²²⁾. The goal of this study was to evaluate a series of synthesized thiosemicarbazide captopril derivatives (2-7)⁽²³⁾ , as ACEI (Fig. 1).

Several methods have been described in the literature for the evaluation of ACE inhibitor activity⁽²⁴⁾; one of them involves UV-VIS spectrophotometry (Beckman DU640). In this method, N- hippuryl-L- histidyl-L-leucine hydrate (HHL), a synthetic substrate is hydrolyzed by ACE to hippuric acid (HA) and N- hippuryl-L- leucine (HL) ⁽²⁵⁾ (Fig. 2). The ability of the synthetic compounds to inhibit the activity of ACE is directly related to the rate of HA formation⁽²⁶⁾. This advanced method offers more simplicity, high sensitivity, cost, and effectiveness because there is no need to separate HA from the reaction mixture.

MATERIALS AND METHODS

Captopril as (s,s) isomer was purchased from Sigma-Aldrich, Germany; ACE from rabbit lung acetone powder, HHL, HA, benzyl sulfonyl chloride (BSC), Triton X-100 and pyridine, were purchased from Sigma-Aldrich (USA).

Preparation of lung ACE

ACE was extracted from rabbit lung acetone powder in the laboratory. The process involved the addition of 2.5g of acetone powder to 25ml of 0.05 M sodium borate buffer, pH8.2 containing 0.3M sodium chloride, and 0.5% Triton X-100 at 4^οC for 16-18h. The extract was then centrifuged at15000 rpm for 60 min at 4^οC. The supernatant layer was dialyzed against the same buffer solution, without Triton X-100; two times, the first with 100 ml for 1h, then with 1000 ml for 24h, and stored at -20^οC until use ⁽²⁷⁾.

Optimization of reaction conditions, pyridine/BSC ratio, temperature, and time

The conditions for the colorimetric reaction of HA and pyridine-BSC were optimized before the ACE inhibitor assay starting.The serial ratio of pyridine to BSC (1:1, 1.5:1, 2:1, 2.5:1, and 3:1), was tested at 150μl of 250 μM HA at 37^οC for 30min to achieve maximum absorbance. The selected ratio was used to optimize the temperature of reaction (25, 30, and 37^οC) at (30, 60, 120, 180, and 240 min).

Calibration curve for HA

A stock solution of HA (1mM) was prepared by dissolving HA in 0.05 M sodium borate buffer (pH8.2), containing 0.3M sodium chloride. Different concentrations were prepared from stock solution by serial dilution with the same buffer over a range of (50-250 μM). To 200μl of each HA prepared concentration, 200μl of (1M) HCl was added, followed by 400μl pyridine then 200μl of BSC. The mixture was mixed by inversion, cooled for 10 min on ice, and then absorbance was measured at 409 nm using a spectrophotometer⁽²⁸⁾.

In vitro colorimetric assay of ACE inhibitor activity

Different concentrations, ranging from 50-250μM of each test compound, were prepared from their corresponding stock solutions using (DMSO: borate buffer (at pH 8.2); 0.5:9.5, and sonicated in a sonicator water bath for 1h. 2.5 mM of HHL substrate was prepared by dissolving 10mg of HHL in 10ml borate buffer. The assay mixture contained (125μl) of test concentration, (50μl) of HHL, and (25μl) of ACE enzyme extract. After incubation at 37^οC for 30min, the reaction was arrested by the addition of (200μl) of (1M) HCl, then (400μl) of pyridine was added, followed by (200μl) of BSC, the mixture was mixed by inversion, and left for 10min, at ice bath. The absorbance of the developed yellow layer was measured at 409nm, using a UV-VIS spectrophotometer. The blank sample was prepared by adding (125μl) of the buffer instead of the inhibitor sample, while a control sample was prepared by adding ACE before the addition of (200μl) of HCl^(29-31). ACE inhibition was expressed as percent inhibition and calculated using the following equation:

Equation 1:

Where: A blank is the absorbance of a blank, A sample is the absorbance of a sample; A control is the absorbance of the control.

Determination of IC₅₀and Ki of captopril and its thiosemicarbazide derivatives

The values of Ki for captopril and its derivatives were calculated from different plot concentrations of inhibitors (50-250μM) against velocity, using Equation 2.While the IC₅₀ value is defined as the concentration of the inhibitor required to decrease the ACE activity by half. This can be calculated using Equation 3.

Equation 2:

Where V, is the velocity without an inhibitor, V_obs is the velocity in the presence of inhibitor at [I] concentration; Km, Michaels Menten constant without inhibitor; [S] the concentration of substrate; Ki, inhibition constant; [I], inhibitor concentration at which V_obs was measured.

Equation 3:

Where Ki, is inhibition constant, [S] the concentration of substrate, and Km, Michaels Menten constant without inhibitor.

Statistics

The data for this study were analyzed using SPSS version 24. The one- way ANOVA test was used to detect the differences between angiotensin-converting enzyme inhibitor activity of captopril and its new derivatives. p values < 0.05 were considered to be significant.

Docking study of captopril and its derivatives

Molecular docking studies were achieved to give insights into the molecular binding modes of the synthesized compounds inside the pocket of the angiotensin-converting enzyme (ACE) using MOE 2015 software. The binding sites were generated from the co-crystallized ligand, captopril, within the angiotensin-converting protein (PDB code: 2c6n).

Water molecules were removed from the complex at first. Subsequently, the crystallographic disorders and unfilled valence atoms were corrected by means of protein report and utility and clean protein option; protein energy was minimized by applying CHARMM and MMFF94 force fields. The rigid part of the binding site was the structure of a protein and was obtained by applying a fixed atom constraint. The protein essential amino acid was defined and prepared for the docking process. The captopril 2D structure and its derivatives (2-7) were drawn using Chem-Bio Draw Ultra14.0. 3D. Structures were protonated, and energy was minimized by applying RMSD of 0.05 kcal/mol. Minimized structures were then prepared for docking using the prepared ligand protocol. The docking process was carried out using the CDOCKER protocol. The receptor was held rigid, while the ligands were allowed to be flexible during the refining process. Each molecule was allowed to produce seven different interaction poses with the protein.

In silico pre ADMET analysis

ADMET prediction was estimated with absorption, distribution, metabolism, and toxicity by the ADMET descriptor module of the small molecules protocol of pre-ADMET online software. These descriptors include human intestinal absorption, the solubility of each compound in the water at 25^οC, blood-brain barrier penetration following oral administration, cytochrome P450 2D6 (CYP2D6) enzyme inhibition, potential liver toxicity, and plasma protein binding.

Physicochemical properties of captopril and its derivatives

The correlation between physicochemical properties of captopril derivatives such as Clog p, topological polar surface area (TPSA), and molecular volume (MV) with their ACE inhibition activity, were calculated using Chem. Informatics on the Web (http://www. molinspiration.com).

RESULTS AND DISCUSSION:

Optimization of the reaction conditions

HA is ordinarily present in urine as a metabolic product. It can react with BSC in the presence of pyridine to form a product (compound A) with a yellow colour that turns red as the concentration of HA increases. This coloured compound shows absorption at (λ_max409nm), using a UV-VIS spectrophotometer (Fig. 3), and (Fig. 4). Higher absorbance of this compound was observed at the pyridine/ BSC ratio of (2:1) at 37^οC over (30 - 180 min). The concentration of HA decreased after 180 min. This refers to the stability of the complex for up to 3h. The minimum time at which higher concentration was obtained is therefore considered to be the preferred reaction time (Fig. 5A and B).

Fig. 4: UV-spectrum of compound A at rang 200-800nm.

Fig. 5: (A) Effect of pyridine/BSC ratio on HA concentration after 30 min at 37^οC. (B) Effect of different combinations of reaction temperatures and time at the pyridine/ BSC ratio (2:1).

Calibration curve

A linear relationship between the range of HA concentrations (50-250μM) and their absorbance at 409nm was obtained, as shown in the following linear regression equation:

Equation 4:

Y=0.0024x+0.0149 R² =0.9969

Where y is the absorbance at λ 409 nm, and x is the concentration of HA.

ACE inhibition assay

Captopril and its derivatives (2-7) inhibitory activity against ACE have been evaluated in vitro using a colorimetric assay method. The principle of this assay depends on the ability of ACE to hydrolyze HHL into HA and HL. The strength of the synthetic compounds to inhibit ACE activity was expressed as a percent inhibition, and showed an inverse correlation with the concentration of HA released. The percent inhibition ± SD and IC₅₀ are mentioned in Table 1. Compounds 4 and 7 showed good ACE inhibition activity compared to captopril, with percent inhibitions of 76.2 ±0.44 and 79.2 ±0.25, and IC₅₀values of 0.137 and 0.103 μM, respectively, while compound 5 showed lower inhibition activity with higher IC₅₀ (46.48 % and 1.45 μM), respectively. This was even better with it, than with captopril. This suggests that it would important for thiosemicarbazide moiety to be involved in new captopril derivatives (2-7), resulting in increased ACE inhibition activity compared to the parent drug. Therefore, the thiosemicarbazide- derivatives substituted at the para-position of the phenyl ring with an electron- withdrawing group displayed more significant activity than the un-substituted ring, while the introduction of an electron-donating group, revealed less improvement in the activity.

The smaller inhibition constant (Ki) refers to a higher binding affinity, and a lower amount of compound needed to inhibit the activity of the enzyme. Compound 7 has a small Ki value (0.05) µM, which is another indicator of good inhibition activity towards ACE.

Table 1: The values of % inhibition and IC₅₀ of Captopril and its derivatives.

Compound No.	INH% ±SD	IC₅₀μM	Ki μM
2	59.19± 0.44	0.291	0.141
3	65.32± 0.25	0.238	0.116
4	76.23± 0.44	0.137	0.066
5	46.48± 0.51	1.45	0.705
6	61.13± 0.25	0.45	0.218
7	79.22± 0.25	0.103	0.050
Captopril (1)	41.55± 0.25	3.70	1.79

% INH: Percent inhibition that calculated as an average of 3 absorbencies for each concentration, p < 0.05; SD: standard deviation; Ki inhibition constant.

Molecular docking

The docking scores (CDOCKER interaction energy) of the best-fitting poses with the active site at the angiotensin-converting enzyme were recorded, as shown in (Table 2). The following parameters were predicted from the docking experiment: the anticipated binding mode, affinity, preferred orientation of each docking pose, and binding free energy (∆G) of the target compounds, and co-crystallized ligand (captopril) with the angiotensin-converting enzyme. The calculated interaction energies for the synthesized compounds (Table 2) were in proper accordance with the experimental results, which organize the compounds in the order of the reduction in the binding energy to ACE, as follows: 7> 4> 3> 2> 6> 5, compared to the other members, and the reference standard. The molecular surface area was calculated, and mapping was performed on the synthesized compounds, and CAP, which showed that the newly synthesized CAP derivatives fit more to the receptor pocket than captopril itself. The literature has documented the critical binding site of ACE, consisting of amino acids Gln259, Ala332, Lys489, Tyr498, Tyr501, and Glu362⁽³²⁾.

The proposed captopril binding mode revealed an affinity value of -4.53 kcal/ mol (Table 2). The captopril carboxylic group exhibited hydrogen bonding interactions with Lys489 and Gln259. The SH group formed additional hydrogen bond interactions with Glu362 and Ala332. The OH of a carboxylic group established an H- arene interaction with Tyr501 (Fig.6, comp. 1).

The anticipated binding mode of compound (2) showed an affinity value of -6.33 kcal/mol. There were two hydrogen bonds between the (C=S, N-H) groups and Ala332, while the C=O group interacted with Tyr501 and His361.In addition, aromatic moiety demonstrated aromatic stacking (pi-interaction) with His361, (Fig.6, comp. 2).

The binding mode of compound (3) displayed an affinity value of -6.45 kcal/mol (Table 2). There were three hydrogen bonds between (C=S, C=O, and SH) groups, and residues of Thr496, His361,and Gln259 amino acids, respectively, while pyrrolidine moiety interacted with His331 by an H-arene interaction (Fig.6, comp.3).

Compound (4) produced binding mode with ACE better than the general pattern observed by captopril. The affinity value is -6.99 kcal /mol. There were three hydrogen bonds between the (SH) group and residues of Lys489, Gln259, Tyr498 amino acids, and two hydrogen bonds between the C=O group interacted with His 491 and His361, while the (C=S) moiety bound to Ala334 (Fig.6,comp. 4).

The expected binding mode of compound (5) demonstrated an affinity value of -4.58 kcal/mol. It offered five hydrogen bonds with residues of Ala334, Ala332, His361, and Tyr501 amino acids (Fig.6.comp. 5).

While the expected binding mode of compound (6) exhibited an affinity value of -5.50 kcal/mol, there was one hydrogen bond between the (C=S) group and residue of Thr496 amino acid. The aromatic moiety was also, demonstrated aromatic stacking (pi-interaction) with Ala334 (Fig.6, comp. 6).

Finally, the binding mode of compound (7) revealed an affinity value of -7.30 kcal/ mol. It displayed four hydrogen bonds between the (CH₂-S, C=O, Cl) groups, and the residues of His491, Gln259, Lys489, and Asp140 amino acids, respectively, (Fig.6,comp. 7).

Table 2: Docking binding energy (Kcal/mol) of the synthesized CAP derivatives, with ACE pocket-forming H-binding and pi- interactions.

Compound No.	Binding free energy (ΔG )Kcal/mol	No. of bonds
Compound No.	Binding free energy (ΔG )Kcal/mol	H.B	pi
2	-6.33	4	1
3	-6.45	3	1
4	-6.99	7	0
5	-4.58	5	0
6	-5.50	1	1
7	-7.30	4	0
Captopril (1)	-4.53	4	1

In silico ADMET analysis

According to the data in (Table 3), the ability of the synthesized compounds to penetrate the BBB has shown to be inadequate. Moreover, it was realized that most of the synthesized compounds possessed appropriate human intestinal absorption compared to captopril. It is well known that many drug candidates have failed during clinical trials ,due to problems related to their absorption properties if HIA value less than 40 ^(33,34).The enhancement of absorption is expected to be due to the aromatic moiety, which increases lipophilicity. By inspecting our compounds, it was established that their aqueous solubility logarithmic level equals to 2 or 3 in most of members, indicating moderate to good aqueous solubility. It is important to indicate that the target compounds would have higher plasma protein binding than captopril. (Table 3).

The cytochrome P450 2D6 model predicts the inhibitory and non-inhibitory behaviour of the chemical structure. It was found that all the titled compounds were found to be non-inhibitors of CYP2D6, despite the fact that captopril has an inhibitory effect on P450 2D6. This indicates that the liver dysfunction effect of the synthetic compounds is not expected upon administration, as shown in (Table 3). Finally, the target compounds are less likely to predict cancer, as seen in (Table 4).

Table 3: Predicted ADMET analysis for the designed compounds (2-7) and reference Captopril.

Compound No.	BBB level	HIA	CYP2D6	PPB	Solubility level
2	0.0590	92.64	0	93.62	3
3	0.121	93.65	0	92.00	2
4	0.112	94.00	0	95.23	2
5	0.143	93.11	0	94.6	2
6	0.167	93.02	0	92.06	3
7	0.338	90.9	0	93.84	2
Captopril (1)	0.239	79.47	Inhibitor	60.47	4

BBB: Blood-Brain Barrier; HIA: Human Intestinal Absorption; CYP2D6: Cytochrome P2D6, 0= non-inhibitor; PPB: Plasma Protein Binding; Solubility level: (4) high sol, (3) and (2) intermediate sol., (1) less sol., (0) poor sol.

Physicochemical properties of captopril and its derivatives

One of the vital physicochemical properties is Clog p, which expresses the degree of lipophilicity of the chemical compound. It is significant to note that the Clog p values for the most active compounds (2-7) ranged from 1.67 to 2.99 These values may explain the difference in their ACE inhibition activity, based on their lipophilicity.

The Clog p values for most compounds lie within the ideal range of lipophilicity⁽³⁵⁾. Based on these results, we noted a correlation between ACE inhibition of the target compounds, and their lipophilic characteristics (Table 5).In addition, the topological polar surface area (TPSA) is another crucial feature associated with drug bioavailability; the passively absorbed molecules with topological polar surface area, more than >140 have low oral bioavailability⁽³⁶⁾. Therefore, the synthesized compounds showed appropriate TPSA values. Moreover, the molecular volume (MV) descriptor regulates the transport characteristics of molecules, such as intestinal absorption. It was observed that the target compounds exhibited good molecular volume values, when compared to captopril. The results are summarized in (Table 5).

Table 5: Clog p, molecular volume and, the molecular polar surface area of Captopril , and its derivatives (2-7).

Compound	Clog p	MV	TPSA
2	1.67	324.11	73.46
3	2.33	341.99	73.50
4	2.27	334.64	73.46
5	2.23	354.47	73.46
6	2.40	357.94	73.06
7	2.99	371.47	74.46
Captopril(1)	0.62	195.65	57.61

CONCLUSION

The colorimetric assay is a simple, fast, and effective method for in vitro assessment of ACE inhibition activity. Compound 7 was the best of the synthesized derivatives, with the highest percent inhibition (79.22±0.25), the lowest IC₅₀ (0.103μM), and the highest binding affinity to ACE with Ki (0.05) µM, compared to the parent CAP drug. There was good correlation with the docking study results, i.e. compound 7 possessed a lower binding energy (-7.30) Kcal/mol, and four H-bonds with active site residues of the enzyme. Pre-ADMET analysis has shown that an increase in the captopril lipophilicity by simply replacing an aromatic ring of thiosemicarbazide derivatives, with an electron- withdrawing group, will improve bioavailability and, subsequently, ACE inhibition activity.

ACKNOWLEDGMENT:

We are grateful to members of the Department of Pharmaceutics, Basrah Pharmacy College –Basrah University, for their support in accomplishing the work. Also, we are extremely thankful to Dr. Mahmood H. Jasim and Dr.Yasser F. Mustafa, College of Pharmacy-Dept of the Pharmaceutical Chemistry/University of Mosul, for their valuable comments and revisions. Many thanks and gratitude to Dr. Abdulrahman Mohammed Saleh, Medicinal Chemistry and drug design MSc. student, Faculty of Pharmacy for Boys – Al-Azhar University - Cairo /Egypt, abdo.saleh240@gmail.com, or biomol.center@gmail.com, for docking analysis,

CONFLICT OF INTEREST

There are no conflicts of interest.

REFERENCES:

1. Bonesi M, Loizzo MR, Statti GA, Michel S, Tillequin F, Menichini F. The synthesis and angiotensin converting enzyme (ACE) inhibitory activity of chalcones and their pyrazole derivatives. Bioorganic and Medicinal Chemistry Letters. 2010;20(6):1990-1993.

2. Kwon M-J, Ahn S-Y. Factors affecting blood pressure control in elderly Koreans with hypertension. Research Journal of Pharmacy and Technology. 2018;11(4):1398-1403.

3. Atlas SA. The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. Journal of managed care pharmacy. 2007;13(8 Supp B):9-20.

4. Muñoz-Durango N, Fuentes CA, Castillo AE, González-Gómez LM, Vecchiola A, Fardella CE. Role of the renin-angiotensin-aldosterone system beyond blood pressure regulation: molecular and cellular mechanisms involved in end-organ damage during arterial hypertension. International journal of molecular sciences. 2016;17(7):2-17.

5. Rekha R, Anju G, Shishant S, Sheefali M, Sanju N. Effect of chemical enhancers on in-vitro permeation of Losartan potassium. Research Journal of Pharmacy and Technology. 2012;5(3): 346-352.

6. Persson PB. Renin: origin, secretion and synthesis. The Journal of Physiology. 2003;552(Pt 3):667-71.

7. Kurtz A. Control of Renin Synthesis and secretion. American Journal of Hypertension. 2012;25(8):839-847.

8. van Sande ME, Scharpé SL, Neels HM, Van Camp KO. Distribution of angiotensin converting enzyme in human tissues. Clinica Chimica Acta. 1985;147(3):255-260.

9. Remuzzi G, Perico N, Macia M, Ruggenenti P. The role of renin-angiotensin-aldosterone system in the progression of chronic kidney disease. Kidney International. 2005;68:S57-S65.

10. Kumar KA, Jagannath P, Saleshier MF. Discovery of novel flavonoid analogues as angiotensin converting enzyme inhibitors based on pharmacophore modelling and virtual screening techniques. Research Journal of Pharmacy and Technology. 2018;11(10):4370-4378.

11. Coates D. The angiotensin converting enzyme (ACE). The international journal of biochemistry and cell biology. 2003;35(6):769-73.

12. Zhao Y, Xu C. Structure and function of angiotensin converting enzyme and its inhibitors. Chinese Journal of Biotechnology. 2008;24(2):171-176.

13. Duchin KL, McKinstry DN, Cohen AI, Migdalof BH. Pharmacokinetics of captopril in healthy subjects and in patients with cardiovascular diseases. Clinical pharmacokinetics. 1988;14(4):241-259.

14. Bahadur S, Chanda R, Roy A, Choudhury A, Das S, Saha S. Preparation and evaluation of mucoadhesive microcapsules of captopril for oral controlled release. Research Journal of Pharmacy and Technology. 2008;1(2):100-105.

15. Lee PW. Handbook of Metabolic Pathways of Xenobiotics: John Wiley and Sons Incorporated; 2014.

16. Migdalof BH, Antonaccio MJ, McKinstry DN, Singhvi SM, Lan SJ, Egli P. Captopril: pharmacology, metabolism and disposition. Drug metabolism reviews. 1984;15(4):841-869.

17. Jaiswal H, Ansari VA, Pandit J, Ahsan F. Pulsatile Drug delivery system: An overview with special emphasis on Losartan and Captopril. Research Journal of Pharmacy and Technology. 2019;12(7):3175-3188.

18. Bevilacqua M, Vago T, Rogolino A, Conci F, Santoli E, Norbiato G. Affinity of angiotensin I-converting enzyme (ACE) inhibitors for N- and C-binding sites of human ACE is different in heart, lung, arteries, and veins. Journal of cardiovascular pharmacology. 1996;28(4):494-499.

19. Attoub S, Gaben AM, Al‐Salam S, Al Sultan M, John A, Nicholls MG. Captopril as a potential inhibitor of lung tumor growth and metastasis. Annals of the New York Academy of Sciences. 2008;1138(1):65-72.

20. Bartosz M, Kedziora J, Bartosz G. Antioxidant and prooxidant properties of captopril and enalapril. Free Radical Biology and Medicine. 1997;23(5):729-735.

21. Skowasch D, Viktor A, Schneider-Schmitt M, Lüderitz B, Nickenig G, Bauriedel G. Differential antiplatelet effects of angiotensin converting enzyme inhibitors. Clinical research in cardiology. 2006;95(4):212-216.

22. Atkinson A, Robertson J. Captopril in the treatment of clinical hypertension and cardiac failure. The Lancet. 1979;314(8147):836-839.

23. Hiba NAS, Ammar A.Razzak Mamood , Redha I.AB. Design, synthesis, docking study and antiplatelet evaluation of new thiosemicarbazide derivatives derived from captopril. Oriental journal of chemistry. 2019;35(2): 829-838.

24. Costa MFdM, Carmona AK, Alves MF, Ryan TM, Davies HM, Anderson GA. Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis. Journal of veterinary science. 2011;12(1):21-25.

25. Islamudin Ahmad AY, Kamarza M, Abdul Mun'im. Review of angiotensin-converting enzyme inhibitory assay: Rapid method in drug discovery of herbal plants. pharmacognasy review. 2017;11(21):1-7.

26. Patil PR, Rakesh SU, Dhabale PN, Burade KB. Simultaneous estimation of ramipril and amlodipine by UV spectrophotometric method. Research Journal of Pharmacy and Technology. 2009;2(2):304-307.

27. Zhuang X-d, Liao L-z, Dong X-b, Hu X, Guo Y, Du Z-m. Design, synthesis, and antihypertensive activity of curcumin-inspired compounds via ace inhibition and vasodilation, along with a bioavailability study for possible benefit in cardiovascular diseases. Drug design, development and therapy. 2016;10:129-140.

28. Jimsheena V, Gowda LR. Colorimetric, high-throughput assay for screening angiotensin I-converting enzyme inhibitors. Analytical chemistry. 2009;81(22):9388-9394.

29. Chen J, Wang Y, Ye R, Wu Y, Xia W. Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme. Food chemistry. 2013;141(4):3329-3334.

30. Sharifi N, Souri E, Ziai SA, Amin G, Amanlou M. Discovery of new angiotensin converting enzyme (ACE) inhibitors from medicinal plants to treat hypertension using an in vitro assay. DARU Journal of Pharmaceutical Sciences. 2013;21(1):1-8.

31. Wu S, Sun J, Tong Z, Lan X, Shu B, Liu Y, et al. Rapid and simple colorimetric assay for screening angiotensin I-converting enzyme inhibitors. Pharmaceutical biology. 2012;50(10):1303-1309.

32. Masuyer G, Douglas RG, Sturrock ED, Acharya KR. Structural basis of Ac-SDKP hydrolysis by angiotensin-I converting enzyme. Scientific reports. 2015;5:1-12.

33. Eissa IH, El-Naggar AM, El-Sattar NE, Youssef AS. Design and discovery of novel quinoxaline derivatives as dual DNA intercalators and topoisomerase II inhibitors. Bentham science. 2018;18(2):195-209.

34. Hardjono S, Widiandani T, Purwanto BT, Nasyanka AL. Molecular docking of N-benzoyl-N’-(4-fluorophenyl) thiourea derivatives as anticancer drug candidate and their ADMET prediction. Research Journal of Pharmacy and Technology. 2019;12(5):2160-2166.

35. Rudraraju A, Amoyaw P, Hubin T, Khan M. Determination of log p values of new cyclen based antimalarial drug leads using RP-HPLC. Die Pharmazie-An International Journal of Pharmaceutical Sciences. 2014;69(9):655-662.

36. Veber DF, Johnson SR, Cheng H-Y, Smith BR, Ward KW, Kopple KD. Molecular properties that influence the oral bioavailability of drug candidates. Journal of medicinal chemistry. 2002;45(12):2615-2623.

Received on 17.10.2019 Modified on 19.11.2019

Research J. Pharm. and Tech 2020; 13(6): 2733-2741.

DOI: 10.5958/0974-360X.2020.00486.2

Compound No.	Ames test	Carcinogen on mouse	Carcinogen on rat	Carcinogenicity	TA100-NA
2	Mutagen	Negative	Negative	0	Negative
3	Mutagen	Negative	Positive	0	Negative
4	Mutagen	Negative	Negative	0	Negative
5	Mutagen	Negative	Negative	0	Negative
6	Mutagen	Negative	Negative	0	Negative
7	Mutagen	Negative	Negative	0	Negative
Captopril (1)	Mutagen	Negative	Negative	0	Negative