Synthesis, structural characterization, and evaluation of new peptidomimetic Schiff bases as potential antithrombotic agents

Abstract

New Schiff bases functionalized with amide and phenolic groups synthesized by the condensation of 2-hydroxybenzaldehyde and 2-hydroxyacetophenone with amino acid amides which in turn were prepared in two steps from N-Boc-amino acids and homoveraltrylamine through intermediate compounds N-Boc-amino acids amides. The compounds were characterized by elemental analysis, FT-IR, UV–Vis, and NMR spectroscopy. The crystal structures of three Schiff bases were determined by single crystal X-ray diffraction. There exists O–H$\cdots$N, N–H$\cdots$O, and C–H$\cdots$O types of hydrogen bonds and C–H$\cdots \mathrm{\pi }$ secondary bonding interactions in these crystalline solids. The Schiff bases have been screened for anticoagulant and antiplatelet aggregation activities. All the compounds showed procoagulant activity which shortens the clotting time of citrated human plasma in both platelet-rich plasma and platelet-poor plasma except the derivatives of L-methionine which showed anticoagulant activity by prolonging the clotting time. In addition, the compounds derived from benzyl cysteine and phenylalanine showed adenosine diphosphate induced antiplatelet aggregation activity, whereas others did not show any role. Moreover, all these compounds revealed non-hemolytic activity with red blood cells.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00706-022-02936-6.

Introduction

Amino acids and proteins are the building blocks of life. Amino acids are neurotransmitters and participate in a number of processes mainly in biosynthesis [1]. Peptides are compounds derived from amino acids which have gained enormous popularity and relevance in the recent years particularly, with the emergence of unnatural analogs as components of molecules with therapeutic potential [2–7]. Many biologically active peptides has been discovered and characterized during the last 30 years [8]. Recently, the replacement of natural amino acids in peptides with non-proteinogenic derivatives to obtain drug-like target molecules has become an important goal in synthetic organic chemistry [9, 10]. One of the interesting areas of research in drug design has been the synthesis of peptidomimetic molecules that are expected to have the same therapeutic effects like natural peptide counterparts with an added advantage of metabolic stability [11, 12].

Dopamine (DA) and their derivatives are active compounds for stress, anxiety, and related behaviors. DA dysfunction found to play a key role in social-anxiety disorders [13–19]. 2-(3,4-Dimethoxyphenyl)ethan-1-amine also known as homoveratrylamine is a dimethyl derivative of DA and is one of the starting material for number of nuerotransmitting agents as well as other biologically important compounds [20–22].

Schiff bases are characterized by an imine (> C=N–) linkage typically obtained by facile condensation of carbonyl compounds (mostly aromatic aldehydes and ketones) with primary amines. Schiff bases have been found versatile ligands as they can form stable complexes with most of the transition metals [23]. Schiff bases found applications in catalysis [24] and material science [25]. Furthermore, Schiff bases and their metal complexes are excellent pharmacophore [26–29] such as antibacterial, antifungal, antiviral, antimalarial, antiinflammatory, antioxidant, anticancer, cytotoxic, enzyme inhibitory therapeutics and including anti-COVID-19 [30–37]. Especially, Schiff bases derived from biologically active starting materials namely isatin [30], 2-azetidinone [31], cephalothin [32], and dopamine [13–16] showed interesting biological activities.

Thrombotic disorders increase the risk of stroke and heart attack. Hence, mortality and morbidity rate have been tremendously increased worldwide [38–43]. The underlying cause involved in the pathophysiology of thrombosis is hyperactivation of platelets and coagulation factors [44, 45]. Known procoagulants for the treatment of haemophilia, anticoagulant (Ex. apixaban, dabigatran, edoxaban, enoxaparin, heparin, rivaroxaban, warfarin) and antiplatelet (Ex. clopidogrel, ticagrelor, prasugrel, dipyridamole, eptifibatide, dipyridamole, aspirin, ticlopidine) drugs for thrombotic disorders, the life-threatening side effects such as internal bleeding, miscarriage, nausea and headache limit their usage [42, 43]. Therefore, identification of the anticoagulant and antiplatelet agents with least side effects is of current interest. Schiff bases based on thiosemicarbazones [46–48], hydrazones [49–53], quercetin, catechol, 1,3-indandione, coumarin [54–58] and several others [59–64] have been reported to be effective antiplatelet aggregation agents. Recently reported that anticoagulants derived from snake venom (Ancrod), fungi aspergillusoryzae (Brinase), sweet clover, saliva of leach and extracts of Flax seeds and Jackfruit seeds [38–43, 65, 66], silver nanoparticles [67, 68] and pyrazinyl pyridine [69].

In this regard, we herein reported the synthesis and characterization of new amide and phenol functionalized Schiff’s bases 13–18 derived from amino acids and homoveraltrylamine. These Schiff’s bases have been explored for antithrombotic and antiplatelet aggregation activities.

Results and discussion

Synthesis

The N-Boc-amino acid amides (2S)-tert-butyl [1-[(3,4-dimethoxyphenethyl)amino]-4-(methylthio)-1-oxobutan-2-yl]carbamate (5), (2S)-tert-butyl [3-(benzylthio)-1-[(3,4-dimethoxyphenethyl)amino]-1-oxopropan-2-yl]carbamate (6), (2S)-tert-butyl [1-[(3,4-dimethoxyphenethyl)amino]-1-oxopropan-2-yl]carbamate (7), and (2R)-tert-butyl [1-[(3,4-dimethoxyphenethyl)amino]-1-oxo-3-phenylpropan-2-yl]carbamate (8) were synthesized by the procedure reported in the literature [70–73] by condensation reaction of 2-(3,4-dimethoxyphenyl)ethan-1-amine with the corresponding N-Boc-(S)-amino acids such as (2S)-2-[(tert-butoxycarbonyl)amino]-4-(methylsulfanyl)butanoic acid (1), (2S)-3-(benzylsulfanyl)-2-[(tert-butoxycarbonyl)amino]propanoic acid (2), (2S)-2-[(tert-butoxycarbonyl)amino]propanoic acid (3), and (2R)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoic acid (4), respectively, in presence of DCC as a coupling agent in DCM at 0 °C. Deprotection of Boc from 5 to 8 with TFA resulted in the corresponding N-[2-(3,4-dimethoxyphenyl)ethyl] functionalized amino acids amides 9–12 such as (2S)-2-amino-N-[2-(3,4-dimethoxyphenyl)ethyl]-4-(methylsulfanyl)butanamide (9), (2S)-2-amino-N-[2-(3,4-dimethoxyphenyl)ethyl]-3-(benzylsulfanyl)propanamide (10), (2S)-2-amino-N-[2-(3,4-dimethoxyphenyl)ethyl]propanamide (11), and (2R)-2-amino-N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenylpropanamide (12). The compounds 5, 7, 8, 9, 11, and 12 were confirmed by determining their melting points which were found in the range of their literature values [70–73].

Schiff’s bases 13–16 such as (S)-N-(3,4-dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]-4-(methylthio)butanamide (13), (S)-3-(benzylthio)-N-(3,4-dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]propanamide (14), (S)-N-(3,4-dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]propanamide (15), (R)-N-(3,4-dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]-3-phenylpropanamide (16), (S)-N-(3,4-dimethyoxyphenylethyl)-2-[[1-(2-hydroxyphenyl)ethylidene]amino]-4-(methylthio)butanamide (17), and (S)-3-(benzylthio)-N-(3,4-dimethyoxyphenylethyl)-2-[[1-(2-hydroxyphenyl)ethylidene]amino]propanamide (18) were obtained by the condensation reaction between amino acids amides 9–12 and 2-hydroxybenzaldehyde taken in equimolar ratio in dry methanol at RT [74–79]. The Schiff bases 17 and 18 were similarly obtained by the reaction of 9 and 10 with 2-hydroxyacetophenone. The reaction mixtures were stirred for 3–4 h. The resulted crude yellow solids on recrystallization in a 1:1 of mixture of chloroform and n-hexane form desired pure crystalline Schiff bases. The equations for the reactions involving the synthesis of compounds 5–18 are shown in Scheme 1. The elemental analysis of 5, 10, and 13–18 agreed with their molecular formulae and confirmed the formation all these compounds.

UV–Vis spectroscopy

UV–Vis spectra (Fig. 1) of the newly synthesized compounds 13–18 have been recorded for their 2.5 × 10^–5 M solutions in DMSO. The intense high-energy absorption bands observed in the range of λ_max = 257–260 and 278–280 nm were assigned to the $\pi$–$\pi$* transitions of the aromatic rings. The low energy and less intense bands appeared between λ_max = 319–321 nm were attributed to the $n-\pi$* transitions within the delocalized $\pi$ system [74–79].

Fig. 1.

UV–Vis spectra of compounds 13–18

FT-IR spectroscopy

In the FT-IR spectra of compounds 6 and 10, the bands observed in the region of ν, 3322 and 3363 cm⁻¹ were due to N–H stretching and the bands appeared in the range $\stackrel{-}{\nu }$  = 2800 to 2900 cm⁻¹ were assigned to aromatic C–H stretching, respectively. In 6, sharp peak appeared at 2100 cm⁻¹ was due to OCONH stretching which indicates Boc (t-Bu) group attached to the amine, which is absent in 10 which indicates its formation. A band appeared at 1684 and 1670 cm⁻¹ in the IR spectra of compounds 6 and 10, respectively, was assigned to CONH stretching. The FT-IR spectra of compounds 13–18 have been compared with the spectra of similar known compounds and the spectra found in agreement with the literature reports [74–79]. The bands observed in the region of 3326–3340 cm⁻¹ attributed to the O–H stretching. The strong bands appeared at 1625–1630 cm⁻¹ were assigned to stretching of the C=N (imine) group present in all these compounds. The stretching bands for amide CONH group observed at around 1650–1655 cm⁻¹. These compounds also displayed bands at 1260 and 1240 cm⁻¹ which were attributed to the C-O stretching. The bands other functionalities appeared at standard wave number regions of their IR spectra. The representative IR spectra of 13–18 are given in Fig. S1 to Fig. S6, respectively (ESI).

NMR spectroscopy

The ¹H NMR spectra of compounds 6, 10, and 13–18 were found characteristic [74–79]. In the ¹H NMR spectra of both 6 and 10, the aromatic protons appeared in range of δ = 6.69 to 7.26 ppm and the methoxy protons appeared as a singlet at 3.84 and 3.83 ppm, respectively. In the compound 6, the NHCO proton observed as a triplet in the range of 6.34–6.37 ppm and Boc [Me₃C] protons appeared as a singlet at 1.45 ppm. While in 10, the NHCO proton shifted downfield and appeared as a broad singlet at 7.38 ppm and the free NH₂ protons appeared as a broad peak at 1.80 ppm.

In the ¹H NMR spectra of Schiff bases 13–18, the signal for phenolic OH proton observed at about 12.2 ppm and was deshielded due to the presence of intramolecular O–H$\cdots$N hydrogen bonding even in solution. In these compounds, the ArCH₂ and CH₂N protons appeared as triplets in the range of 2.74–2.77 and 3.46–3.59 ppm, respectively. The methoxy protons observed as independent singlets at 3.81 and 3.78 ppm, respectively. The signal for azomethine (HC=N) proton observed as a singlet in the range of 7.90–8.35 ppm in all the compounds 13–18. The amide proton (CONH) appeared as a triplet at 6.0–6.1 ppm in 13–18 on coupling with neighboring CH₂ protons. The signals for aromatic protons appeared in the region 6.6–7.3 ppm. The representative ¹H NMR spectra of 6, 10, and 13–18 are given in Fig. S7 to Fig. S25, respectively (ESI).

The ¹³C{¹H} NMR spectra compounds 6, 10, and 13–18 were found characteristic [74–79]. In the ¹³C NMR spectra of 6 and 10, the peak for CONH carbon appeared at δ = 175 and 177 ppm, respectively, and the peaks for methoxy (OCH₃) carbons appeared at ~ 60 ppm. In 6, the signal for Boc carbonyl carbon appeared at 159.9 ppm. In the ¹³C NMR spectra of 13–18, the signal for ArCH₂ carbon appeared at 34.4 ± 0.2 ppm whereas the signal for NCH₂ carbon found deshielded and observed at about 40.4 ppm. The peaks for phenolate (ArCO⁻), azomethine (C=N), and amide (CONH) carbons appeared at around 160, 166, and 170 ppm, respectively, in all these Schiff base compounds. The peaks for both OCH₃ carbons appeared independently at slightly different chemical shifts at around 55.3 ± 0.2 ppm in all these compounds. The observations from NMR spectra of 13–18 confirmed their formation. The representative ¹³C{¹H} NMR spectra of compounds 13–18 are given in Fig. S26 to Fig. S32, respectively (ESI).

Mass spectrometry

The amide functionalized Schiff bases 13–18 were characterized by mass spectrometry using electro spray ionization in combined with liquid chromatography (ESI-LC–MS) method. The ESI-LC–MS spectra of 13–18 showed molecular ion (M⁺) peaks at m/z = 417, 479, 357, 432, 431, and 492.9, respectively, and were agreed with their calculated molecular weights. Hence, mass spectral data confirmed the formation of the respective compounds 13–18. The representative mass spectra of compounds 13–18 are given in Fig. S33 to Fig. S38 (ESI).

Single-crystal X-ray diffraction

The single-crystal X-ray diffraction data for 13, 15, and 17 collected and their molecular structures in solid state were determined. The selected crystal data and structure refinement parameters of 13, 15, and 17 are given in Table 1 (complete data is given in Table S1 in ESI) and their selected bond parameters are given in Table 2. The molecular structures of the compounds 13, 15, and 17 as ORTEP diagrams are shown in Figs. 2, 3, and 4, respectively. The compounds 13, 15, and 17 crystallize in monoclinic system and non-centrosymmetric P2₁ space group with Z = 2. All the molecules consist of a chiral carbon atom with S-configuration and the two molecules differ in conformations of the two chains connected to these chiral carbons. There exists an intramolecular O–H$\cdots$N hydrogen bond in each compound which is a characteristic for 2-hydroxyaryl Schiff bases [80].

Table 1.

Selected crystal data and structure refinement parameters for 13, 15, and 17

Compound	13	15	17
Empirical formula	C22H28N2O4S	C20H24N2O4	C23H30N2O4S
Formula weight/g mol−1	416.52	356.42	430.55
Crystal system	Monoclinic	Monoclinic	Monoclinic
Space group, Z	P21, 2	P21, 2	P21, 2
a/Å	8.482(3)	9.692(2)	4.9379(3)
b/Å	5.1415(15)	5.1081(8)	14.2499(9)
c/Å	24.614(7)	19.469(4)	15.9154(9)
α, β, γ/°	90, 99.92, 90	90, 102.77(2), 90	90, 94.354(5), 90
Volume/Å3	1057.4(5)	940.0(3)	1116.65(12)
ρcalc/g cm−3	1.308	1.2591	1.281
μ/mm−1	0.184	0.088	0.176
F (000)	444.0	380.2	460
Reflections collected	7980	3686	3966
Independent reflections	3533 [Rint = 0.1003, Rsigma = 0.2199]	2643 [Rint = 0.0160, Rsigma = 0.0690]	2920 [Rint = 0.0129]
Data/restraints/parameters	3533/1/267	2643/3/238	2920/30/299
GOF on F2	0.921	0.994	1.098
Final R indexes [I >  = 2σ (I)]	R1 = 0.0572, wR2 = 0.0999	R1 = 0.0419, wR2 = 0.0647	R1 = 0.0365, wR2 = 0.0676
Final R indexes [all data]	R1 = 0.1306, wR2 = 0.1169	R1 = 0.0947, wR2 = 0.0767	R1 = 0.0521, wR2 = 0.0732
CCDC No	1,999,894	1,999,895	2,031,920

Table 2.

Selected bond lengths and bond angles of 13, 15, and 17

13		15		17
Bond lengths/Å
S(0AA)-C(23)	1.803 (5)	–	–	S(1)-C(20)	1.791(4)
S(0AA)-C(24)	1.810 (5)	–	–	S(1)-C(19)	1.801(4)
C(10)-O(25)	1.231(6)	O(2)-C(9)	1.234(4)	O(2)-C(9)	1.224(3)
C(19)-O(21)	1.354 (5)	O(1)-C(1)	1.344(4)	O(1)-C(1)	1.346(4)
N(12)-C(13)	1.294 (6)	N(1)-C(7)	1.270(4)	N(1)-C(7)	1.280(4)
N(12)-C(11)	1.460 (6)	N(1)-C(8)	1.462(4)	N(1)-C(8)	1.469(4)
N(9)-C(10)	1.339 (6)	N(2)-C(9)	1.332(4)	N(2)-C(9)	1.324(4)
N(9)-C(8)	1.447 (5)	N(2)-C(10)	1.462(4)	N(2)-C(10)	1.474(8)
–	–	–	–	N(2)-C(10')	1.487(8)
Bond angles/°
C(14)-C(13)-N(12)	122.9(4)	C(6)-C(7)-N(1)	122.1(3)	C(6)-C(7)-N(1)	116.8(3)
H(13)-C(13)-N(12)	118.5(0)	H(7)-C(7)-N(1)	118.95(2)	C(21)-C(7)-N(1)	125.1(4)
C(13)-N(12)-C(11)	114.9 (4)	C(7)-N(1)-C(8)	118.0(3)	C(7)-N(1)-C(8)	121.8(3)
C(10)-N(9)-C(8)	123.4 (4)	C(9)-N(2)-C(10)	123.4(3)	C(9)-N(2)-C(10)	124.7(4)
–	–	–	–	C(9)-N(2)-C(10')	117.2(4)
C(23)-S(0AA)-C(24)	98.2 (3)	–	–	C(20)-S(1)-C(19)	100.4(2)

Fig. 2.

ORTEP view of 13 with thermal ellipsoids drawn at 30% probability. Intramolecular O–H$\cdots$N hydrogen bond is shown as thin dashed lines

Fig. 3.

ORTEP view of 15 with thermal ellipsoids drawn at 30% probability. Intramolecular O–H$\cdots$N hydrogen bond is shown as thin dashed lines

Fig. 4.

ORTEP view of 17 with thermal ellipsoids drawn at 30% probability. Intramolecular O–H$\cdots$N hydrogen bond is shown as thin dashed lines

The crystal structure of 13 possess N9-H9$\cdots$O25 hydrogen bonded C4 chains linking the molecules along b-axis (Fig. 5, Table 3). The crystal structure of 15 similarly features N2-H2N$\cdots$O2 intermolecular hydrogen bonds linking the molecules into C4 chains running parallel to b-axis. These adjacent parallel chains in 17 are further interlinked via C19-H19C$\cdots$π and C20-H20B$\cdots$π interactions to form a one-dimensional ribbon parallel to b-axis (Fig. 6, Table 3). Contrary to 15, in 13, the adjacent parallel chains are further interlinked through C24-H24C$\cdots$O21 intermolecular interactions running as C11 chains parallel to a-axis, thereby, forming a two-dimensional (2D) sheet parallel to the ab-plane. Therefore, different architectures are observed in both the crystal structures.

Fig. 5.

A partial view of the crystal packing displaying two-dimensional sheets in 13 through NH$\cdots$O and CH$\cdots$O hydrogen bonding

Table 3.

Geometric parameters for hydrogen bonds and other intermolecular contacts (Å,°) operating in the crystal structures of 13, 15, and 17

	D-H$\cdots$A	D-H	H$\cdots$A	D$\cdots$A	D-H$\cdots$A
13	O21-H21$\cdots$N12#	0.82	1.94	2.6086	139
	N9-H9$\cdots$O25i	0.86	2.18	3.0084	162
	C24-H24C$\cdots$O21ii	0.96	2.56	3.3292	137
15	O1-H1O$\cdots$N1#	0.84	1.86	2.568(4)	141
	N2-H2N$\cdots$O2v	0.86	2.32	3.165(4)	171
	C19-H19C$\cdots$πv,a	0.96	2.75	3.610(5)	149
	C20-H20B$\cdots$πvi,a	0.96	2.88	3.717(4)	147
17	O1-H1O$\cdots$N1#	0.85(4)	1.77(4)	2.531(4)	147(4)
	N2-H2N$\cdots$O2iii	0.85(2)	2.10(2)	2.940(3)	174(4)
	C11-H11A$\cdots$O4iv	0.97	2.59	3.371(10)	137

^#Intra; i: x, 1 + y, z; ii: 1 + x, − 1 + y, z; iii: − 1 + x,y,z; iv: –x,–1/2 + y,1–z; v: x, –1 + y, z; vi: 1–x, 1/2 + y, 1–z; a:Centroid of C12-C17 aromatic ring

Fig. 6.

A partial view of the crystal packing displaying ribbon-like framework in 15 formed via NH$\cdots$O hydrogen bonds and CH$\cdots$π interactions

The crystal structure of 17 also features N2-H2$\cdots$O2 intermolecular hydrogen bonds linking the molecules into C4 chains running parallel to a-axis similar to that observed in the other two structures. The adjacent parallel chains are further interlinked through C11-H11A$\cdots$O4 intermolecular interactions running as C7 chains parallel to b-axis, thereby, forming a two-dimensional (2D) sheet parallel to the ab-plane (Fig. 7, Table 3). The crystal structures of 13 and 17 are similar, except for the variation of the length of C-H $\cdots$ O chains in the two structures: C11 in 13 and C7 in 17.

Fig. 7.

A partial view of the crystal packing displaying two-dimensional sheets in 17 through NH$\cdots$O and CH$\cdots$O hydrogen bonding

The dihedral angle between the two aromatic rings is 85.0(1)° in 13, 85.74(18)° in 17, and 89.2(2)° in 15. The two methoxy groups and the attached aromatic ring in 13 are in the same plane with the torsions C27-C3-O2-C1 and C5-C4-O1-C18 having values of 1.3(7)° and − 2.0(8)°. The corresponding torsions C13-C14-O3-C22 and C16-C15-O4-C23 in 17 have values of 4.0(6)° and 2.8(6)°, respectively, while, in 15, torsions C13-C14-O3-C19 and C16-C15-O4-C20 have values of 5.8(5)° and − 10.4(5)°, respectively, indicating that the two methoxy groups are in the same plane as that of the attached ring. The –CH₂–CH₂– groups in all the three molecules have staggered conformation and the molecular conformations of both the molecules are stabilized by intramolecular O–H$\cdots$N hydrogen bonds between azomethine N and phenolic H closing into a S(6) motif. In 17, the ethylene –CH₂–CH₂– group is disordered over the C–C bond in the ratio 0.5:0.5.

Role of compounds 13–18 in blood coagulation cascade

To check the probable therapeutic potency on thrombotic disorders, the newly synthesized Schiff bases 13–18 were scrutinized for their possible role in blood coagulation cascade and platelet function. The plasma re-calcification time experiment briefly involved that the compounds 13–18 (0 to 40 µg) were initially pre-incubated with 0.1 cm³ of citrated human platelets rich plasma (PRP) in presence of 10 mM Tris–HCl (10 mm³) buffer pH 7.4 for 5 min at 37 °C. Then, 20 mm³ of 0.25 M CaCl₂ was added to the pre-incubated mixture and the clotting time was recorded in s. Plots were drawn for clotting time against amount of compounds (µg) and are shown in Fig. 8.

Fig. 8.

Plasma re-calcification time of 13–18 using human plasma as PRP (pink) and PPP (green)

All the compounds exhibited an effect on coagulation cascade in both PRP and PPP which increases with increasing the amount of any compound. Among the compounds screened, the Schiff bases of methionine with 2-hydroxybezaldehyde (13) and 2-hydroxyacetophenone (17) showed an anticoagulation effect by extending the clotting time from control 210 ± 3.5 to 270 ± 1.6, 210 ± 3.5 to 315 ± 1.5 s in PRP and 221 ± 1.5 to 283 ± 2.5, 220 ± 1.3 to 325 ± 1.6 s in PPP, respectively, at a maximum amount of 40 µg of any compound as represented in Fig. 9 when compared with a standard drug heparin (1 µM) as a positive control. Contrary to the above, the Schiff bases derived from benzyl cysteine (14 and 17), alanine (15) and phenyl alanine (16) showed procoagulant effect by reducing the clotting time from the control 189 ± 0.2 to 68 ± 3, 221 ± 1.6 to 122 ± 1.3, 243 ± 1.5 to 155 ± 1.2 and 210 ± 1.2 to 60 ± 3 s in PRP and 200 ± 3.5 to 80 ± 0.9, 230 ± 1.6 to 131 ± 1.3, 246 ± 1.5 to 160, and 220 ± 2.5 to 62 ± 0.3 s in PPP, respectively. Furthermore, the salicyladehyde Schiff bases of benzyl cysteine (14) and phenylalanine (16) showed strong procoagulant activity as revealed by their magnitude of decrease in clotting time from control in both PRP and PPP by about 120 and 160 s than that of alanine (15) which exhibited 90 s respectively. While procoagulant activity of the Schiff base 17 of 2-hydroxy acetophenone with benzyl cysteine showed 100 s which is about 20 s less than the Schiff base 14 of salicylaldehyde. But, in case of methionine, the Schiff base of 2-hydroxyacetophenone showed an anticoagulant activity by increment of 100 s in both PRP and PPP than that of the salicyladehyde Schiff base which showed 60 s.

Fig. 9.

Effect of compounds 13–18 in blood coagulation cascade at a maximum amount of 40 µg

Role of compounds 13–18 in platelet aggregation function

Platelets are anucleated cells which possess numerous membrane-bound receptors for physiological agonists such as collagen, ADP, thrombin, epinephrine, arachidonic acid (AA) and platelet activating factor [49–53]. Binding of an agonist to the receptors alter the platelet morphology leading to their activation and aggregation. Thus, platelet activation/aggregation plays a key role in the pathophysiology of thrombotic disorders [43]. Therefore, the new amide and phenol functionalized Schiff base compounds 13–18 were examined for their interference in the platelet function using PRP and PPP in the presence and absence of an agonist. The experiment was performed on an aggregometer connected with a pen recorder. As platelets aggregate, light transmission increases progressively producing a trace on the recorder which would be the plot of light transmission between PRP and PPP corresponds to the 0 and 100% aggregation respectively against time in s. The pre-incubated compounds 13–18 (10 to 30 µg) with 0.45 cm³ PRP or PPP for 3 min were added ADP (5 µM), an agonist and the aggregation was followed for 6 min. Curiously, among the compounds tested, the Schiff bases derived from benzyl cysteine (14 and 18) and phenylalanine (16) augmented the platelet aggregation induced by a potent agonist ADP as shown in Figs. 10b, 11b, and 12b, respectively. While, the compounds synthesized from methionine (13 and 17) and alanine (15) did not interfere at all in agonist induced platelet function. The percentage of platelet activation by the compounds 14, 16, and 18 was found to be 29, 41, and 45%, respectively. These observations demonstrated that the compounds 14, 16, and 18 derived from benzyl cysteine and phenylalanine containing an aromatic and bulkier group at chiral carbon exhibited strong procoagulant property and augmentation of platelet activation.

Fig. 10.

a Activation of ADP (5 µM) induced platelet aggregation without (Trace 1) and with increasing amount (10–30 µg) of compound 14 (Traces 3, 5, and 7) and b dose (10–30 µg)-dependent aggregation (bar graph) by compound 14

Fig. 11.

a Activation of ADP (5 μM) induced platelet aggregation without (Trace 1) and with increasing amount (10–30 µg) of compound 16 (Traces 3, 5, and 7) and b dose (10–30 µg)-dependent aggregation (bar graph) by compound 16

Fig. 12.

a Activation of ADP (5 μM) induced platelet aggregation without (Trace 1) and with increasing amount (10–30 µg) of compound 18 (Traces 3, 5, and 7) and b dose (10–30 µg)-dependent aggregation (bar graph) by compound 18

Non-hemolytic activity of compounds 13–18

To identify the probable interaction of synthesized compounds on red blood cell (RBC) membrane, hemolytic assay was carried out using RBCs. Strikingly, all the compounds incapable of lysing the RBC membrane, as intact RBCs were identified in compounds treated with RBC sample, while, a positive control water caused haemolysis and PBS buffer as a negative control which as shown in Fig. 13.

Fig. 13.

Hemolytic activity of compounds 13–18 measured at 540 nm against water (positive control) and PBS buffer (negative control)

Conclusions

Six new chiral Schiff bases functionalized with peptide and phenol groups 13–18 have been synthesized and characterized by FT-IR, UV–Vis, ¹H and ¹³C{¹H} NMR spectroscopy, and LC–MS. The crystal structures of three Schiff bases were determined by single crystal X-ray diffraction. These crystalline solids, the molecules are found to associate by moderate to strong OH$\cdots$N intramolecular, N–H$\cdots$O, and C–H⋯O types of intermolecular hydrogen bonds as well as C–H⋯π intermolecular secondary bonding interactions in 15 forming the supramolecular assemblies. These compounds have been demonstrated to interfere in blood coagulation cascade and platelet aggregation function. Among which benzyl cysteine (14 and 18), alanine (15), and phenylalanine (16)-based Schiff bases exhibited procoagulant activity, whereas methionine (13)-based compound showed a mild anticoagulant activity. Surprisingly, among all the compounds tested, only benzyl cysteine (14 and 18) and phenylalanine (16)-based Schiff bases showed platelet activating potency. These compounds also showed non-hemolytic activity towards RBCs. Thus, these compounds may serve as better prototype in identifying new drugs with least side effects. Thus, it requires further conformational studies in this prospective. Therefore, understanding of their mechanism of action on coagulation cascade and platelet aggregation function may provide more insight into therapeutic usage of said compounds in the management of thrombotic disorders.

Experimental

2-(3,4-Dimethoxyphenyl)ethan-1-amine (97%), N,N’-dicyclohexyl carbodiimide (DCC) (99%), 2-hydroxyacetophenone (99%), N-Boc-L-methionine, and N-Boc-L-benzylcysteine were purchased from Sigma Aldrich, USA. 2-Hydroxybenzaldehyde (99%), N-Boc-L-alanine and N-Boc-L-phenylalanine were obtained from Spectrochem Ind. Pvt. Ltd. Solvents such as methanol (MeOH), petroleum ether (40–60 °C), dichloromethane (DCM), chloroform (CHCl₃), n-hexane, diethylether, and other chemicals of commercial analytical reagent grade were purchased from Merck India Ltd., and used as received. Melting points were determined in open capillary tubes closed at one end using a RAGA melting point apparatus. Elemental analysis (C, H, and N) was performed on a LECO–CHNSO–9320 type elemental analyzer. UV–Vis absorption spectra were recorded in ethanol using Agilent Cary-60 spectrophotometer in the spectral window of 200–800 nm. FT-IR spectra were recorded on a PerkinElmer Frontier MIR/FIR spectrometer using ATR. ¹H and ¹³C{¹H} NMR spectra were recorded on Agilent VNMRS-400 (6 and 10) and Bruker WM-400 (13–18) 400 MHz NMR spectrometers using tetramethylsilane (TMS) as an internal standard. Single-crystal X-ray diffraction data were collected on Bruker APEX2 (13) and Oxford Diffraction Xcalibur (TM) (15 and 17) single crystal X-ray diffractometers with CCD detector. The structures were solved by SHELXT [81] and refined using SHELXL [82] available within Olex software [83, 84]. The compound 15 is a weak anomalous scatterer. We feel that the presented structural information on this compound is reliable enough to unambiguously solve and refine the structure reliably. In 17, though Friedel Pair Coverage is low, the Flack parameter is enough and presented structural information on this compound is reliable in order to unambiguously solve and refine the structure reliably. Thermo Scientific BioMate-6 UV–Visible spectrophotometer was used for determination of haemolytic assay. Lumi aggregation system of Model-700 was used for the determination of platelet aggregation. Blood samples were collected from healthy voluntary donors.

(S)-tert-Butyl [3-(benzylthio)-1-[(3,4-dimethoxyphenethyl)amino]-1-oxopropan-2-yl]carbamate (6, C₂₅H₃₄N₂O₅S)

A mixture of 10 cm³ dry CH₂Cl₂, 1000 mg N-Boc-L-benzylcysteine (2, 3.2 mmol), 582 mg 2-(3,4-dimethoxyphenyl)ethan-1-amine (3.2 mmol), and 0.662 mg DCC (3.2 mmol) was maintained for 3 h at 0 °C in an ice bath with constant stirring. The reaction was monitored by TLC using a mixture of petroleum ether and ethyl acetate as an eluent. The mixture was then filtered to separate the solid N,N’-dicyclohexyl urea formed from DCC and was discarded. The filtrate was evaporated under reduced pressure on a rotary evaporator. The resulting oily mass was treated with 20 cm³ anhydrous ice-cooled acetone to separate additional N,N’-dicyclohexyl urea. The acetone solution was evaporated under reduced pressure and the resulting crude product was purified by crystallization from n-hexane and chloroform. White solid; yield: 920 mg (60%); m.p.: 160–162 °C; FT-IR (ATR): $\stackrel{-}{\nu }$ = 3322, 2932, 2856, 2108, 1684, 1654, 1513, 1450, 1239, 1150, 1012, 840, 814, 698 cm⁻¹; ¹H NMR (399.82 MHz, CDCl₃): δ = 7.26–7.29 (m, 3H, H21,22), 7.20–7.23 (m, 1H, H23), 6.76–6.78 (d, ³ J = 8.4 Hz, 1H, H7), 6.69–6.71 (d, ³ J = 7.2 Hz, 2H, H11,10), 6.34–6.37 (t, 1H, NH), 5.27 (s, 1H, NH-Boc), 4.16–4.17 (b, 1H, CH), 3.84 (s, 3H, OCH₃), 3.83 (s, 1H, OCH₃), 3.69–3.70 (d, ² J = 1.9 Hz, 2H, SCH₂Ph), 3.44–3.50 (q, 2H, NCH₂), 2.80–2.85 (dd, ³ J = 13.99 Hz, ² J = 5.6 Hz, 1H, CH₂S), 2.71–2.75 (t, 2H, CH₂Ar), 2.67–2.70 (dd, ³ J = 13.99 Hz, ² J = 5.6 Hz, 1H, CH₂S), 1.41 (s, 9H, t-Bu) ppm; ¹³C{¹H} NMR (99.995 MHz, CDCl₃): δ = 175.1 (C2), 159.9 (t-BuOCO), 153.7 (C7), 152.4 (C8), 142.52 (C20), 135.7 (C5), 133.6 (C21), 133.2 (C22), 131.8 (C23), 125.3 (C10), 116.5 (C6), 116.0 (C9), 84.9 (t-BuC), 60.5 (OCH₃), 60.3 (OCH₃), 58.4 (C1), 41.1 (C3), 39.8 (C4), 38.5 (C19), 32.9 (C18), 30.2 (t-Bu), 29.6 (t-Bu), 29.3 (t-Bu) ppm.

(2S)-2-Amino-N-[2-(3,4-dimethoxyphenyl)ethyl]-3-(benzylsulfanyl)propanamide (10, C₂₀H₂₆N₂O₃S)

The compound 6 (500 mg, 1.10 mmol) in 5 cm³ dry CH₂Cl₂ taken in a round bottom flask and this solution was stirred for 10 min at around 0 °C using an ice-bath. A solution of 5 cm³ trifluroacetic acid (TFA) in 5 cm³ CH₂Cl₂ was added to the above solution. The mixture was allowed to react maintaining the above temperature under continuous stirring further for 4 h. The reaction was monitored by TLC using ethyl acetate and petroleum ether (50:50) as an eluent. At the completion of the reaction, the mixture was added drop-wise a saturated aqueous solution of NaHCO₃ until the pH 8 reached. The organic phase separated and was treated with solid anhydrous Na₂SO₄ to eliminate water residues, filtered, and evaporated under reduced pressure. Yellow oily liquid; yield: 355 mg (90%); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3363, 2938, 2850, 2838, 1670, 1519, 1451, 1231, 1143, 1026, 814, 765, 697 cm⁻¹; ¹H NMR (399.82 MHz, CDCl₃): δ = 7.38 (bs, 1H, NH), 7.26–7.29 (m, 4H, H21,22), 7.207–7.23 (m, 1H, H23), 6.76–6.78 (d, ³ J = 8.4 Hz, 1H, H6), 6.69–6.73 (m, 2H, H9,10), 3.82 (s, 6H, OCH₃), 3.67 (s, 2H, SCH₂Ph), 3.42–3.48 (q, 2H, NCH₂), 3.36–3.39 (dd, ³ J = 14.0 Hz, ² J = 4.0 Hz, 1H, CH), 2.90–2.95 (dd, ³ J = 13.8 Hz, ² J = 3.9 Hz, 1H, CH₂S), 2.71–2.74 (t, 2H, CH₂Ar), 2.58–2.63 (dd, ³ J = 13.6 Hz, ² J = 8.4 Hz, 1H, CH₂S), 1.77–1.80 (bs, 2H, NH₂) ppm; ¹³C{¹H} NMR (99.995 MHz, CDCl₃): δ = 177.8 (C2), 153.6 (C7), 152.3 (C8), 142.7 (C20), 136.0 (C5), 133.6 (C21), 133.5 (C21’), 133.2 (C22), 131.8 (C23), 125.3 (C6), 116.6 (C10), 116.0 (C9), 60.5 (OCH₃), 60.3 (OCH₃), 58.5 (C1), 41.9 (C3), 41.0 (C4), 35.5 (C19), 35.5 (C18) ppm.

Synthesis of Schiff bases of amides of amino acids (13–16)

The compound 9/10/11/12 (256/306/206/269 mg, 1.0 mmol) was dissolved in 20 cm³ dry methanol and the resulting solution was stirred for 0.5 h at room temperature (RT). 2-Hydroxybenzaldehyde (100 mg, 1 mmol) in 20 cm³ dry methanol was added to the above solution drop-wise with stirring. The mixture was stirred further for 2–3 h at RT during which time the solution changes to yellow. The progress of the reaction was monitored on TLC using petroleum-ether and ethyl acetate as a mobile phase. After completion, the reaction solution was concentrated on a rotary evaporator to result a yellow precipitate. The yellow precipitate was washed twice with n-hexane (10 cm³ × 2) and then dried under vacuum. The dry solid was recrystallized from a mixture of chloroform and n-hexane (1:1) resulting yellow crystalline solids of 13/14/15/16, respectively.

(S)-N-(3,4-Dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]-4-(methylthio)butanamide (13, C₂₂H₂₈N₂O₄S)

Yellow crystalline solid; yield: 325 mg (95.3%); m.p.: 87–89 °C;$[{\propto ]}_D^{24}=-24.{69}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3340, 2930, 2850, 1650, 1631, 1550, 1513, 1260, 1235, 1140, 1030, 809, 760, 640 cm⁻¹; UV–Vis: λmax (ε) = 260 (17,972), 279 (13,294), 321 (4910) nm (M⁻¹ cm⁻¹); ¹H NMR (399.65 MHz, CDCl₃): δ = 12.30 (s, 1H, OH), 8.35 (s, 1H, CH = N), 7.38–7.42 (td, 1H, H16), 7.29–7.31 (dd, ² J = 1.6 Hz, ³ J = 8.0 Hz, 1H, H17), 6.99–7.02 (d, ³ J = 8.0 Hz, 1H, H14), 6.94–6.97 (td, 1H, H15), 6.61–6.67 (m, 3H, H6, 9,10), 6.05 (bs, 1H, CONH), 4.04–4.07 (dd, ³ J = 9.2 Hz, ² J = 4.0 Hz,1H, CH), 3.82 (s, 1H, OCH₃), 3.78 (s, 1H, OCH₃), 3.45–3.59 (m, 2H, NCH₂), 2.73–2.77 (t, ³ J = 6.8 Hz, 2H, CH₂Ar), 2.51–2.58 (m, 1H, CH₂), 2.29–2.42 (m, 2H, CH₂S), 2.09–2.14 (m, 1H, CH₂), 2.08 (s, 3H, SCH₃) ppm; ¹³C{¹H} NMR (99.99 MHz, CDCl₃): δ = 169.9 (C2), 166.7 (C11), 160.1 (C13), 148.6 (C7), 147.2 (C8), 132.7 (C15), 131.9 (C17), 131.7 (C5), 120.5 (C12), 118.8 (C10), 118.7 (C16), 116.5 (C14), 112.4 (C6), 111.7 (C9), 70.7 (C1) 55.5 (OCH₃), 55.3 (OCH₃), 40.3 (C3), 34.5 (C4), 29.5 (C19), 24.5 (C18), 14.5 (CH₃) ppm; LC–MS (ESI): m/z calcd. 416.53 (M⁺), found 417.

(S)-3-(Benzylthio)-N-(3,4-dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]propanamide (14, C₂₇H₃₀N₂O₄S)

Yellow solid; yield: 370 mg (94.6%); m.p.: 80–83 °C;$[{\propto ]}_D^{24}=-71.{09}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3340, 2925, 2835, 1655, 1630, 1543, 1510, 1255, 1226, 1132, 1030, 804, 755, 640, 530 cm⁻¹; UV–Vis: λmax (ε) = 258 (24,398), 277 (20,756), 321 (6,390) nm (M⁻¹ cm⁻¹); ¹H NMR (399.65 MHz, CDCl₃): δ = 12.20 (s, 1H, OH), 8.16 (s, 1H, CH = N), 7.40–7.42 (td, 1H, H16), 7.25–7.38 (m, 5H, H21, 22), 7.22–7.24 (m, 1H, H17), 6.70–7.02 (d, ³ J = 8.0 Hz, 1H, H14), 6.94–6.97 (t, ³ J = 7.6 Hz, 1H, H15), 6.63–6.67 (m, 3H, H6, 9,10), 6.11–6.14 (s, 1H, CONH), 4.08 (bs, 1H, CH), 3.81 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.68–3.69 (m, 2H, SCH₂Ph), 3.41–3.60 (m, 2H, NCH₂), 3.16–3.20 (dd, ² J = 13.6 Hz, ³ J = 3.6 Hz, 1H, CH₂S), 2.79–2.85 (dd, ² J = 14.0 Hz, ³ J = 8.8 Hz, 1H, CH₂S), 2.73–2.76 (t, ³ J = 6.8 Hz, 2H, CH₂Ar) ppm; ¹³C{¹H} NMR (CDCl₃): δ = 169.1 (C2), 166.9 (C11), 160.1 (C13), 156.6 (C20), 148.6 (C7), 147.2 (C8), 138.3 (C5), 132.7 (C12), 131.8 (C15), 131.7 (C17), 128.8 (C21), 128.3 (C22), 126.8 (C23), 120.5 (C10), 118.8 (C12), 116.5 (C14), 112.8 (C6), 111.8 (C9), 71.3 (C1), 55.4 (OCH₃), 55.3 (OCH₃), 40.5 (C3), 34.4 (C4), 33.3 (C20), 25.3 (C18) ppm; LC–MS (ESI): m/z calcd. 478.6 (M⁺), found 479.

(S)-N-(3,4-Dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]propanamide (15, C₂₀H₂₄N₂O₄)

Yellow crystalline solid; yield: 282 mg (97%); m.p.: 80–82 °C;$[{\propto ]}_D^{24}=-12.{72}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3343, 2981, 2927, 2869, 1650, 1630, 1579, 1542, 1522, 1459, 1419, 1267, 1229, 1138, 1026, 873, 804, 763, 635, 572 cm⁻¹; UV–Vis: λ_max (ε) = 257 (13,788), 278 (11,800), 319 (3,876) nm (M⁻¹ cm⁻¹); ¹H NMR (399.65 MHz, CDCl₃): δ = 12.36 (s, 1H, OH), 8.34 (s, 1H, CH = N), 7.39–7.41 (td, 1H, H16), 7.27–7.30 (d, ³ J = 8.0 Hz, 1H, H17), 6.99–7.01 (d, ³ J = 8.0 Hz, 1H, H14), 6.92–6.96 (t, ³ J = 7.2 Hz, 1H, H15), 6.65–6.68 (m, 3H, H6, 9, 10), 6.06 (s, 1H, CONH), 3.95–4.00 (q, 1H, CH), 3.82 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.44–3.56 (m, 2H, NCH₂), 2.74–2.77 (t, ³ J = 7.2 Hz, 2H, CH₂Ar), 1.52–1.54 (d, ³ J = 3.4 Hz, 3H, CH₃) ppm; ¹³C{¹H} NMR (CDCl₃): δ = 170.9 (C2), 165.8 (C11), 160.3 (C13), 148.6 (C7), 147.2 (C8), 132.5 (C5), 131.8 (C15), 131.8 (C17), 120.5 (C12), 118.9 (C16), 118.7 (C14), 116.8 (C10), 112.4 (C6), 111.7 (C9), 66.6 (C1), 55.4 (OCH₃), 54.9 (OCH₃), 40.4 (C3), 34.6 (C4), 19.7 (CH₃) ppm; LC–MS (ESI): m/z calcd. 356.41 (M⁺), found 357.

(R)-N-(3,4-Dimethyoxyphenylethyl)-2-[(2-hydroxybenzylidene)amino]-3-phenylpropanamide (16, C₂₆H₂₈N₂O₄)

Yellow solid; yield: 340 mg (96.0%); m.p.: 89–90 °C;$[{\propto ]}_D^{24}=-10.{97}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3326, 2923, 2850, 1655, 1625, 1516, 1451, 1237, 1143, 1019, 805, 760, 705, 636, 540 cm⁻¹; UV–Vis: λ_max (ε) = 259 (24,000), 280 (15,420), 319 (6,840) nm (M⁻¹ cm⁻¹); ¹H NMR (399.65 MHz, DMSO-d₆): δ = 12.21 (s, 1H, OH), 7.89 (s, 1H, CH = N), 7.33–7.35 (td, 1H, H16), 7.24–7.22 (d, ³ J = 7.6 Hz, 1H, H17), 7.26–7.09 (m, 5H, H20-23), 6.96–6.99 (d, ³ J = 8.4 Hz, 1H, H14), 6.85–6.89 (t, ³ J = 7.6 Hz, 1H, H15), 6.63–6.65 (m, 2H, H9,10), 6.57–6.58 (m, 1H, H6), 6.01 (bs, 1H, CONH), 3.99–4.02 (dd, ³ J = 8.8 Hz, ³ J = 4.0 Hz, 1H, CH), 3.81 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.464–3.59 (m, 2H, NCH₂), 3.35–3.40 (dd, ³ J = 13.6 Hz, ² J = 3.4 Hz, 1H, H18), 3.06–3.11 (dd, ³ J = 13.2 Hz, ² J = 8.8 Hz, 1H, H18), 2.63–2.76 (m, 2H, CH₂Ar) ppm; LC–MS (ESI): m/z calcd. 432.5 (M⁺), found 432.

Synthesis of Schiff bases of amides of amino acids (17 and 18)

Compound 9/10 (229/275 mg, 0.73 mmol) was dissolved in 20 cm³ dry methanol and stirred the solution for 0.5 h at room temperature (RT). A solution of 100 mg 2-hydroxyacetophenone (0.73 mmol) in 20 cm³ dry methanol was added to the above solution drop-wise under continuous stirring. The resulting mixture was stirred further for 2–3 h at RT during which time the solution changes to yellow. The progress of the reaction was monitored on TLC using petroleum-ether and ethyl acetate (50:50) as mobile phase. The yellow precipitate left, on evaporation of solvent by a rotary evaporator, was washed twice with n-hexane (10 cm³ × 2) and then dried under vacuum. The dry solid was recrystallized from a mixture of chloroform and n-hexane (1:1) resulting a yellow crystalline solids of 17/18, respectively.

(S)-N-(3,4-Dimethyoxyphenylethyl)-2-[[1-(2-hydroxyphenyl)ethylidene]amino]-4-(methylthio)butanamide (17, C₂₂H₂₈N₂O₄S)

Yellow crystalline solid; yield: 284 mg (90%); m.p.: 91–93 °C;$[{\propto ]}_D^{24}=-28.{62}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3304, 3063, 2930, 2850, 1644, 1619, 1543, 1516, 1465, 1452, 1374, 1307, 1236, 1158, 1091, 1028, 855, 803, 752, 675, 562, 524, 429 cm⁻¹; UV–Vis: λ_max (ε) = 280 (14,250), 326 (5.109) nm (M⁻¹ cm⁻¹); ¹H NMR (399.82 MHz, DMSO-d₆): δ = 14.99 (s, 1H, OH), 7.55–7.57 (d, ³ J = 7.6 Hz, 1H, H17), 7.35–7.39 (t, ³ J = 7.6 Hz, 1H, H16), 6.97–6.99 (d, ³ J = 8.0 Hz, 1H, H14), 6.87–6.91 (t, ³ J = 7.2 Hz, 1H, H15), 6.54–6.64 (m, 3H, H6, 9, 10), 5.90 (s, 1H, CONH), 4.52–4.55 (q, 1H, CH), 3.81 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.47–3.52 (q, 2H, NCH₂), 2.71–2.75 (q, 2H, CH₂S), 2.57–2.63 (m, 1H, CHCH₂S), 2.41–2.47 (m, 1H, CHCH₂S), 2.34–2.40 (m, 2H, CH₂Ar), 2.31 (s, 3H, SCH₃), 2.09 (s, 3H, CH₃) ppm; ¹³C{¹H} NMR (99.99 MHz, CDCl₃): δ = 173.1 (C9), 169.9 (C2), 162.7 (C7), 148.6 (C14), 147.2 (C15), 132.4 (C4), 131.7 (C6), 128.9 (C12), 120.5 (C1), 119.3 (C17), 117.7 (C5), 117.1 (C3), 112.5 (C13), 111.8 (C16), 61.6 (C8), 55.5 (OCH₃), 55.3 (OCH₃), 34.4 (C10), 33.2 (C11), 29.6 (C19), 24.4 (C18), 14.9 (SCH₃), 14.5 (CH₃) ppm; LC–MS (ESI): m/z calcd. 430.56 (M⁺), found 431.

(S)-3-(Benzylthio)-N-(3,4-dimethyoxyphenylethyl)-2-[[1-(2-hydroxyphenyl)ethylidene]amino]propanamide (18, C₂₈H₃₂N₂O₄S)

Yellow solid; yield: 329 mg (91%); m.p.: 95–96 °C;$[{\propto ]}_D^{24}=-82.{08}^{\circ }$(c = 0.1, CHCl₃); FT-IR (ATR): $\stackrel{-}{\nu }$ = 3281, 2960, 2837, 1651, 1610, 1552, 1537, 1516, 1504, 1438, 1413, 1330, 1261, 1192, 1136, 1118, 1028, 910, 812, 756, 706 cm⁻¹; UV–Vis: λ_max (ε) = 279 (20,823), 322 (7,400) nm (M⁻¹ cm⁻¹); ¹H NMR (399.82 MHz, DMSO-d₆): δ = 15.49 (s, 1H, OH), 8.042 (s, 1H, H6), 7.62–7.64 (d, 1H, H9), 7.20–7.28 (m, 5H, Ph), 6.72–6.80 (m, 4H, H14, H15, H16, H17), 6.63–6.65 (d, ³ J = 6.4 Hz, 2H, H10), 6.63–6.65 (d, 1H, H12), 5.52–5.54 (s, 1H, CONH), 4.48–4.51 (t, ³ J = 6.4 Hz, 1H, CH), 3.70–3.74 (m, 2H, SCH₂Ph), 3.67 (s, 3H, OCH₃), 3.66 (s, 3H, OCH₃), 3.27–3.30 (m, 2H, NCH₂), 2.67–2.74 (dd, ² J = 13.2 Hz, ³ J = 7.6 Hz, 1H, CH₂S), 2.69–2.72 (dd, ² J = 13.2 Hz, ³ J = 5.6 Hz, 1H, CH₂S), 2.61–2.65 (t, 2H, CH₂Ar), 2.70 (s, 3H, CH₃) ppm; ¹³C{¹H} NMR (99.99 MHz, DMSO-d₆): δ = 173.2 (C9), 169.3 (C2), 162.3 (C7), 156.6 (C20), 148.6 (C14), 147.2 (C15), 138.3 (C12), 132.5 (C1), 131.7 (C4), 129.1 (C6), 128.5 (C21, C25), 128.4 (C22, C24), 126.8 (C23), 120.5 (C17), 117.7 (C5), 117.4 (C3), 112.5 (C13), 111.8 (C16), 62.5 (C8), 55.6 (OCH₃), 55.3 (OCH₃), 35.5 (C10), 34.8 (C11), 33.4 (C19), 24.7 (C18), 15.2 (CH₃) ppm; LC–MS (ESI): m/z calcd. 492.63 (M⁺), found 492.9.

Preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP)

The platelet concentration of PRP was adjusted to 3.1 × 10⁸ platelets/cm³ with PPP. The PRP maintained at 37 °C and was used within 2 h for the aggregation process. All the above preparations were carried out using plastic wares or siliconized glass wares.

Plasma re-calcification time

The plasma re-calcification time experiment was carried out according to the reported method [67–69]. Briefly, the compounds 13–18 (2.5 to 40 µM) were pre-incubated with 0.1 cm³ of citrated human PRP in presence of 10 mM Tris–HCl (10 mm³) buffer of pH 7.4 for 5 min at 37 °C. 20 mm³ of 0.25 M CaCl₂ was added to the pre-incubated mixture and then the clotting time was recorded in seconds.

Platelets’ aggregation

The platelets’ aggregation experiment was carried out by turbidometric method [67–69] using a Chronolog dual channel aggregometer connected to an Omni scribedual pen recorder to record the light transmission as a function of time. The 500 mm³ of reaction volume containing PRP (0.45 cm³) and newly synthesized compounds 13–18 (10 to 30 µM) was pre-incubated for 3 min in a cylindrical glass cuvette under constant stirring. The aggregation was initiated by the addition of adenosine diphosphate (ADP, 5 µM) an agonist and followed for 6 min. Furthermore, these new Schiff bases were checked for their effect on platelet aggregation function in the absence of any agonist. As platelets aggregate, light transmission increases progressively producing an aggregation trace on the recorder which was a plot of light transmission between PRP and PPP corresponding to the 0 and 100% aggregation against time in s.

Non-haemolytic activity

The effect of new Schiff bases 13–18 on red blood cells was studied as per the previously described procedure [67–69]. Hemolytic activity was determined using washed human erythrocytes. Briefly, packed human erythrocytes and phosphate buffered saline (PBS) (1:9 v/v) were mixed. 1 cm³ of this suspension was incubated independently with the various amounts of Schiff bases (0–100 µg) for 1 h at 37 °C. The reaction was stopped by adding 9 cm³ of ice-cold PBS and then centrifuged at 1000 g for 10 min at 37 °C. The amount of hemoglobin released in the supernatant was measured at 540 nm on UV–Vis spectrophotometer. The activity was expressed as percent of haemolysis against 100% lysis of cells due to addition of water that served as a positive control and PBS served as a negative control.

Supplementary Information

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