Structural details on the interaction of biologically active sulfur-containing monoterpenoids with lipid membranes

Journal Pre-proof Structural details on the interaction of biologically active sulfurcontaining monoterpenoids with lipid membranes

Liliya E. Nikitina, Roman S. Pavelyev, Valeriya A. Startseva, Sergei V. Kiselev, Leisan F. Galiullina, Oksana V. Aganova, Ayzira F. Timerova, Sergei V. Boichuk, Zulfiya R. Azizova, Vladimir V. Klochkov, Daniel Huster, Ilya A. Khodov, Holger A. Scheidt PII:

S0167-7322(19)35994-X

DOI:

https://doi.org/10.1016/j.molliq.2019.112366

Reference:

MOLLIQ 112366

To appear in:

Journal of Molecular Liquids

Received date:

30 October 2019

Revised date:

11 December 2019

Accepted date:

20 December 2019

Please cite this article as: L.E. Nikitina, R.S. Pavelyev, V.A. Startseva, et al., Structural details on the interaction of biologically active sulfur-containing monoterpenoids with lipid membranes, Journal of Molecular Liquids(2019), https://doi.org/10.1016/ j.molliq.2019.112366

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© 2019 Published by Elsevier.

Journal Pre-proof Structural details on the interaction of biologically active sulfur-containing monoterpenoids with lipid membranes Liliya E. Nikitina*1,2, Roman S. Pavelyev2, Valeriya A. Startseva1,2, Sergei V. Kiselev1, Leisan F. Galiullina2, Oksana V. Aganova2, Ayzira F. Timerova2, Sergei V. Boichuk1, Zulfiya R. Azizova1, Vladimir V. Klochkov2, Daniel Huster3, Ilya A. Khodov*2,4, Holger A. Scheidt*3 1

Kazan State Medical University, Kazan, Russia

Institute for Medical Physics and Biophysics, Leipzig University, D-04107 Leipzig, Germany 4

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3

Kazan Federal University, Kazan, Russia

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2

G. A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, 153045

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Ivanovo, Russia

KEYWORDS: thioterpenoids, isoborneol, coagulation activity, platelets aggregation, 1D and

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2D solution-state NMR, solid-state NMR, model cell membranes, molecular mechanism of

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coagulation activity

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Journal Pre-proof ABSTRACT. In this work, we propose the synthesis of new thioterpenoids of a bornane series and study the influence of these compounds on hemostasis. The results from this study suggest that among all studied terpenoids, sodium ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2yl]thio) acetate may be the most promising for further research due to enhanced inhibition of the spontaneous aggregation compared with isoborneol, and due to its higher solubility in water compared with ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio) acetic acid, which has approximately the same antiaggregatory and anticoagulant properties. In accordance with one hypothesis, the distribution of the studied bioactive compound molecules within the cellular lipid

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membrane can directly influence the anticoagulant properties. In the current work the interactions of thioterpenoids with phospholipid membranes have been studied using various

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NMR techniques. The findings of this study indicate that sodium ([(1R,2R,4R)-1,7,7-

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trimethylbicyclo[2.2.1]hept-2-yl]thio) acetate, has a membrane location, which is shifted somewhat in the direction of the membrane-water interface, which shields the hydrophobic

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interactions of coagulation factors with the surface of the membrane. Apparently, the absence of

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such properties for isoborneol is caused by its large immersion inside the membrane. These results are an excellent initial step towards creating new drugs based on the synthesized thioterpenoids in order to increase the effectiveness of treatment and prevention of many human

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diseases accompanying disorders in the hemostasis system.

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Journal Pre-proof INTRODUCTION Cardiovascular diseases, which development is caused by the atherosclerotic lesion of the arterial blood vessels, remain the main mortality cause worldwide. The atherosclerosis caused changes of the vessel wall favour the hemostasis activation, which leads to the thrombus formation, which in its turn greatly reduce or even block the blood circulation1–4. Thrombus formation is initiated by the reorganization of the cell membranes (platelets foremost), contacting with blood, which leads to their adhesion, aggregation and activation of the coagulative factors of clotting. Pharmaceuticals, suppressing the platelets activity, are utilized for the purpose of cardiovascular

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diseases treatment and prevention. However, existing nowadays products do not guarantee the efficient prevention or treatment of the disease. Besides, some patients (from 6 to 60 %), taking,

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for example, acetylsalicylic acid, demonstrate tolerance to it5. This explains the researcher’s

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interest in the search and development of new drug compounds, capable to inhibit the platelets activity. Previously we developed synthetic approaches for obtaining sulfur-containing anti-inflammatory,

antimicrobial,

antihelicobacter,

antiaggregatory

and

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antifungal,

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monoterpenoids of different structure6. It was shown that the designed compounds possess anticoagulative activity, have low toxicity, and also are free from the mutagenic and genotoxic effects7–15.

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In this study, we present the synthesis of new thioterpenoids of a bornane series (2-4) and investigate the influence of these compounds on hemostasis. We made the assumption that it is

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the bornane skeleton of thioterpenoids that will be more effective in terms of stabilizing

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phospholipid membranes compared to the pinane and camphene structures studied earlier, because it is conformationally rigid and can be more suitable for anchoring in the lipid bilayer. Dodecylphosphocholine (DPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) micelles have been used for NMR measurements.

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Chemical test.

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METHODS AND MATERIALS

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Compounds purification was performed with the use of column chromatography on a silica gel (60–200 mesh, Acros Organics, USA).

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The development of the reaction and compounds purity was monitored by the analytical thinlayer chromatography (TLC) on Sorbfil PTLC-AF-A-UF plates. Isoborneol, thioglycolic acid and camphene were supplied by Sigma Aldrich Rus. Сompound 2 was prepared within the framework of previously discussed method with some omissible adjustments.16 ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio)acetic acid (2) The mercaptoacetic acid in the amount of 0.01 mol and ВF3.Et2O in the catalytic amount were added to the room temperature solution of 0.01 mol of the (+/-)-camphene1 in 0.01 LofEt2O (see Scheme 1). The process lasted for 600-900 minutes, and thenwe removed the solvent at low pressure. We purified the products of the reaction by means of column chromatography, eluting with the hexane-diethyl ether mixture or solely hexane, on silica gel. The yield of compound 2 was about 67 %.

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Journal Pre-proof Sodium ([(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl]thio)acetate (3) Saturated sodium carbonate solution with great care was mixed with a solution of 0.25 g (0.0011 mol) of compound 2 in methyl alcohol (0.03 L) until gas bubbles ceased, and we stirred the reaction mixture at the room temperature for 60 minutes. Further, we removed the solvent in vacuum. The yield of compound 3 was quantitative. Biochemical assays. The coagulative ability of the designed compounds was established by determining

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spontaneous platelet aggregation and the plasma coagulation activity of specimen from the venous blood of sufferers with coronary heart disease (CHD), with marked changes in a

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hemostatic system. As a control, platelet aggregation was investigated in platelet rich plasma

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taken from healthy individuals (from 21 to 31 years old), who for at least 7–10 days did not take any nonsteroidal antiinflammatory drugs (NSAID’s), aspirin or alternative medicines, which are

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considered to demonstrate an influence on the clotting factors or platelet function. All donors

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were notifiedon these experiments and gave their consent to the study. The Kazan State Medical University’s Ethical Committee gave an approval before any of these experiments started. We performed all processes according to the accepted guidelines. All participants of the experiments

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gave their written permissionin conjunction with the Declaration of Helsinki. Activity of the platelets aggregation was assessed by the Chrono-Log Platelet Aggregometer

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(Chrono-Log Corporation, Pennsylvania, USA) within the G. Born technique.17 For such case,

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we used plasma, taken from the venous blood of the sufferer with an observable hypocoagulation shift in the hemostasis system and those with the ischemic heart disease (IHD). The induced aggregation of platelets was examined on the plasma, donated by the healthy donors (The Center of Emergency Hospital, Kazan City Hospital N 7). 50μL 30 % ethyl alcohol solution containing 0.02 M of tested substance was mixed with 450 μL platelet-rich plasma (PRP), further we incubated this mixture at 37 °C for 5 minutes. In check test, 30 % ethyl alcohol mixture utilized in the compound preparation was added to the plasma. Induced platelet aggregation was defined with the aid of the following solutions: collagen (2 μg / ml), ristotsetin (1 mg / ml), epinephrine (10 μM), adenosine pyrophosphate (5 μM) and arachidonic acid (0.5 mM). The exact pure plasma volume was chosen as the standard for the optical control. The rate of the platelet aggregation was quantified by the maximum value optical density incidence before and after the

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Journal Pre-proof reaction. Comparison of the produced compound with the acetylsalicylic acid allowed us to estimate the relative efficiency of the former, as the latter is considered to be the most significant antiaggregation agent. In this regard, we used the plasma of the sufferers with IHD, who were taking aspirin. Coagulation activity was assessed using an automated coagulometric analyser ACL Top 500 (Instrumentation Laboratory USA). Activity of Coagulation was assessed with the help of Minilab-7001 coagulation rate analyzer (Russia) according to the data of surface-dependent standard tests on coagulation: activated partial thromboplastin time (APTT), prothrombin and thrombin time (TT). Fibrin formation rate

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was evaluated on the Trombodynamic Recorder T-2 device with the video footage of the fibrin clot growth in the space when clot formation was activated from the surface with fixed tissue

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factor.

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We used automatic cytopheresis (Haemonetics corporation MSC+, USA) to obtain platelet concentrate from the stabilized by sodium citrate healthy donors blood.

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The LDP protocol was followed by collecting platelets with poor leukocytes count (not above

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1 × 106 / cells per dose), in a dosage set by the operator (2 × 1011 / cell per dose). We carried out the platelets cytapheresis within the intermittent flow into a chamber through separating system. For the storage of the platelet concentrate we used special cell-saver MSC+ (Haemonetics Corp,

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USA). There it was kept for five days at temperatures from 22 to 24 °С under the constant stirring with the a thrombomixer (Presvak, Argentina). We stabilized platelet concentrate with

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the acid citrate dextrose (ACD) solution as anticoagulant at a 1:9 ratio, consisting of 22 g

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trisodium citrate dihydrate, 24.5 g of dextrose monohydrate, 8 g citric acid monohydrate and dihydrogen oxide all the way to 1 L. We inserted the platelet concentrate samples into the test Vacutainer TM tubes (Becton Dickinson Vacutainer Systems, UK), the amount of microvesicles was estimated using a BD FACSCanto RUO flow cytometer (Becton Dickinson Vacutainer Systems, UK) after the mixing appropriate amounts with phosphate buffer. We counted the total number of microvesicles in 0.001 mL using the light diffusion for 1 minute with the BD CellQuest Pro software (BD Biosciences)6. We then centrifuged the platelet concentrate samples for 20 min at 2.500 g, then supernatant fluid in the amount of 100 μL was added to 900 μL of healthy blood plasma. We prepared this plasma with low quantity of the microvesicles using disc membranes (HA, Millipore, USA) with a pore size of 0.1 μm. Micelles from sodium dodecylphosphocholine micelles (DPC) were prepared according to the recommendations of

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Journal Pre-proof Caillon et al.,18 and their coagulation activity was established in the APTT test, using 40 mM solution of micelles instead of kaolin-cephaline mixture. It was compared with those of DPC and control plasma. Cytotoxicity of the compound 3 was assessed by the MTS-based assay (Promega, Madison, WI) with the minor modifications19. In short, human BJ foreskin fibroblasts (АТСС, USA) were cultured into ultra-low attachment the 96-well plates (Corning Inc., USA) and allowed to grow for one day in DMEM/199 (chase medium) with a supplementation of levoglutamide (0.3 g/l), 10 % of fetal bovine serum (Gibco BRL, USA) and agrimycin-penicillin antibiotics (Russia).

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The cells were cultured at temperature of 37 °C of 5.0 % of carbon dioxide with the compound 3 indicated concentrations, 1.5 % solution of ethanol used as a diluent for compound 3 (control 1),

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whereas the chemotherapeutic agent etoposide (Calbiochem Corp., USA), which was used as a

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positive control (control 2) for cytotoxicity. At last, we added MTS reagent to the cell culture medium to estimate the cell viability. We incubated the MTS reagent with live cells for more

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than 60 minutes and analyzed at 492 nm using a microplate spectrophotometer Multiscan FC

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(Thermo Scientific, USA). Whole data was normalized to the number of live cell, cultured with

Statistical analysis.

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the diluent for compound 3 (control 1).

Software, Inc., USA).

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We carried out all operations with the help of Graph Pad Prismsoftware V.6 (GraphPad

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The obtained data was analyzed using the Kruskal-Wallis and Kolmogorov-Smirnov tests. The results were shown as the average data points and their standard error. The comparative analysis was carried out with the paired sample Student's t-test application. The discrepancies were admitted to be statistical significant at value p < 0.05. Solid and solution state NMR spectroscopy. For solution-state NMR, n-dodecylphosphocholine (SDPC, Sigma-Aldrich Rus) was utilized without further purification. Compound 4 was mixed in pure D2O solvent and DPC/D2O system at concentrations of 22.7 and 18.3 mM, respectively. Compound 3 was mixed in pure D2O solvent and DPC/D2O system in the amount of 3.0 and 12.1 mM, respectively. Compound 2 was dissolved in CDCl3 and DPC/D2O at concentrations of 10.2 and 12.1 mM, respectively. Micelles containing solutions were prepared by use of a mixture of undeuterated and deuterated (>98%)

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Journal Pre-proof DPC. Concentrations of DPC in deuterium oxide were higher than the critical micelle concentrations and corresponded to 13.6 mM for the D-DPC and 9.5 mM for the non-deuterated DPC in case of compound 4; 17.0 mM for the D-DPC and 4.3 mM for the non-deuterated DPC in case of compound 3; 15.0 mM for the D-DPC and 3.2 mM for the non-deuterated DPC in case of compound 2. Required amounts of DPC were weighted for the preparation of the stock solutions in water. After the solubilization of surfactant in deuterium oxide, the base solutions were vortex-mixed and subsequently sonicated using a digital ultrasound cleaner for 5 or 6 minutes. Following the preparation of the stock solution, required amount of the respective

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compounds were admixed, samples were vortex-mixed and then left for the equilibration for the night at the room temperature. (POPC)

or

1-palmitoyl(d31)-2-oleoyl-sn-glycero-3-

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oleoyl-sn-glycero-3-phosphocholine

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For solid-state NMR measurements, the compounds and the phospholipids 1-palmitoyl-2phosphocholine (D-POPC) were mixed in trichloromethane. The solvent was vaporized and the

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resulting lipid film was again dissolved in the tentatively 500 μl of hexamethylene, then frozen in

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liquid nitrogen, and lyophilized under vacuum (~ 150 μbar). 50 wt % of the lipid powder was obtained through the hydration with the deuterium-depleted H2O for static

31

P, static 2H and

magic-angle spinning (MAS) 1H NMR experiments, correspondingly. A homogeneous mixture

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of the lipid suspension was prepared through the twelve cycles of the freeze and thawing and careful centrifugation. Then, we put the lipid samples into the 4-mm MAS rotor for the 1H

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NOESY NMR experiments or 5-mm short glass tubes (handmade) for the static NMR scheme.

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Solution-state NMR experiments were carried out on the Bruker AVANCE III HD NMR spectrometer operating at 700 MHz (1H) frequency equipped with a 5 mm probe, employing standard Bruker TOPSPIN-NMR software V.3.1. No sample spinning was utilized within these experiments. The 1H chemical shifts were referenced by the solvent protons (1H in D2O - 4.78 ppm; 1H in CDCl3 - 7.26 ppm). The data for the proton NMR experiments were obtained with 32.768 complex data points. The spectral characterization of the compounds 1H and

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C NMR signals were obtained from

the chemical shifts, integral values, multiplicities signal from the both type NMR experiments 1

H-13C heteronuclear correlations in 2D HMBC and 2D HSQC spectra, and 1H-1H homonuclear

correlations in 2D COSY spectra.

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Journal Pre-proof Every 2D experiment was performed with 2048 × 512 data points; the sweep widths and the number of transients were individually optimized. The 2D COSY20 and gradient-enhanced 2D NOESY21 NMR experiments were carried out with the technique of the pulsed filtered gradient22. The relaxation delay was chosen to be 2 s, and the π/2 pulse length was 8.0 μs. Duration of mixing time was taken as 400, 300, 200, and 100 ms. The resulting multiple free induction decays (FIDs) for all two dimensional NMR experiments were apodized with the usual sine function and in both dimensions antecedent to performing forward Fourier transformation. The heteronuclear two-dimensional NMR spectra were written down zero-filled to a 2048 × 512

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data matrix and apodized in two dimensions with the standard sine function with a shift. The experiments on the gradient-selected HSQC using phase sensitive Echo/Antiecho-TPPI (Time-

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Proportional Phase Incrementation) gradient selection with decoupling during acquisition, and

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shaped pulse for the uniform inversion of the second channel (13C), optimized for 1J(CH) = 145 Hz. The gradient-selected 1H-13C HMBC NMR experiments23 were carried out with 2J(CH), J(CH) set up to 7 Hz. Pseudo- 2D DOSY experiments were carried out through the stimulated

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3

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spin-echo pulse sequence with bipolar pulse longitudinal eddy current delay (BPPLED)24–28. The pulse gradients length was enhanced for each diffusion delay in an attempt to obtain 1-5 % residual signal at 95 % of the maximum of gradient strength. 31

P NMR procedures for the solid state were carried out on a Bruker DRX300

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Static 2H and

NMR wide-bore spectrometer operating at 121.5 MHz for 31P and at 46.07 MHz for 2H with an

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application of a single-layer solenoid coil with the inner diameter of 5 mm. The deuterium 2H

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NMR spectra were accumulated using the quadrupolar echo sequence29 with a relaxation delay time of 1 s and a spectral width of 100 kHz. Two pulses with 0.0032 ms and π/2 were splitted up by a 0.05 ms delay time. The deuterium 2H NMR powder spectral data were de-Paked30, using the McCabe and Wassall31 algorithm, detailed in32. Smoothed profiles of the order parameter were found from the quadrupolar splittings (ΔνQ), according to the expression33:

( ) (1) where ΔνQ is the experimental quadrupolar splitting, e2qQ/h is the quadrupolar coupling constant, which for the deuterons in C–2H bonds is equal to 167 kHz34, and S(n) is the order parameter of the chain for acyl chains nth carbon of the phospholipids. We used the mean torque model for the calculation of acyl chain length Lc∗35.

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Journal Pre-proof The static

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P NMR solid-state spectra were accumulated using the pulse sequence of Hahn-

echo36 with a 90° pulse length of 3 μs, a spectral width of 500 kHz, the echo delay of 50 μs and a relaxation delay of 2.5 s. Low power broadband proton decoupling was used during static

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NMR experiments. The anisotropy of the chemical shift (Δσ) was determined from numerical simulations of the

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P spectral line shapes with the aid of a code, developed in Mathcad 2001

Professional® (Cambridge, UK). We obtained proton magic angle spinning NMR spectra at a spinning frequency of 6 kHz on a Bruker Avance III 600 MHz high-field NMR spectrometer utilizing a 4-mm high-resolution HR-

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MAS probe equipped with a gradient and 2H lock. Standard 90° pulse lengths were 4 μs. We obtained two-dimensional proton MAS nuclear Overhauser effect spectroscopy (NOESY)

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spectra at the following mixing times: 0.1, 100, 200, 300 and 500 ms37,38. For the most part, 280

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data points were acquired in the indirect dimension with 32 scans per increment at a relaxation time delay of 3.2 s. All solid-state NMR experiments were performed at temperature 30 degree

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We derived the NOESY cross-relaxation rates σij, fitting the experimental NOE buildup curves of the cross-peak volumes at different durations of mixing within the nonlinear regression curve model39:

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fitter in Origin (OriginLab Cooperation, Northampton, MA), as in the equation for the spin pair

  AII (0) 1  exp 2 IStm  exp  tm  2  T1,IS 

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AIS t m  

(2)

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where Aij(tm) and Ajj(0) are the cross-peak volumes at mixing time tmand zero, respectively. T1,ij is the relaxation time, which is inversely proportional to the magnetization leakage to the lattice. RESULTS AND DISCUSSION The sulfide 2 synthesized in our study was obtained by the electrophilic addition reaction of mercaptoacetic acid to racemic camphene 1 in the presence of Lewis acid16 in the catalytic amounts. The reaction proceeded according to Markovnikov’s rule and was accompanied by isomerization of the camphenic structure of the molecule to the bornane structure (Scheme 1). In order to increase the solubility of compound 2, considering the importance of this property for biochemical tests, it was converted to the salt form – compound 3.

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HSCH2COOH

9

7

10

1 6

11

2

o

BF3 * Et2O, 20 C 5

H

4

1

O

12

SCH2C

OH

SCH2C

NaHCO3

H

O ONa

3

3

2 Scheme 1. Synthesis of compounds 2, 3.

Primarily, the effect of these compounds on the platelet activity and plasma coagulation factors

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were studied. Previously, we had shown that terpenes without any other functional groups,

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except for double bonds, do not affect hemostasis12–15. Considering these results and the fact that the synthesis of compound 2 is accompanied by the rearrangement of the initial skeleton of the

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molecule into the bornane structure by analogy with compounds 2 and 3. We did not study camphene 1 but the well-known terpene alcohol isoborneol (compound 4), which structure is

4

5

3

1

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6

9

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similiar to the terpene fragments 2, 3 (Fig. 1).

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2

OH H

4

Figure 1. Chemical structure and three-dimensional isodensity surface of the electrical potential of compound 4. The 1H and

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C NMR signals of compounds 2-4 in solution were identified using two-

dimentional 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC NMR experiments (see Figs. 1S-9S in Supplementary material). The proton and carbon NMR chemical shifts values of compounds 2-4 signals are presented in the Table 1.

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Journal Pre-proof Table 1. 1H and 13C NMR chemical shifts for compounds 3, 4 in D2O and compound 2 in CDCl3 solution Compound 2

Compound 3

Compound 4

δC / ppm

δH / ppm

δC / ppm

δH / ppm

δC / ppm

C-1

-

47.31

-

46.73

-

47.62

CH-2

2.89

55.31

2.67

54.47

4.08

77.49

CH2-3

1.91; 1.96

40.47

1.66; 1.79

39.65

0.97; 2.33

37.56

CH-4

1.76

46.07

1.58

45.50

1.69

44.74

CH2-5

1.19; 1.75

27.27

1.01; 1.55

26.58

1.25; 1.80

27.70

CH2-6

1.22; 1.71

38.54

1.06; 1.53

37.83

1.35; 1.79

25.78

C-7

-

49.82

-

48.88

-

48.80

CH3-8

0.99

20.33

19.82

0.92

19.46

CH3-9

0.86

20.00

0.69

19.43

0.91

17.98

CH3-10

1.04

13.50

0.84

13.00

0.89

17.75

CH2-11

3.29

35.80

3.06

39.06

-

-

C-12

-

175.95

-

179.00

-

-

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δH / ppm

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Atoms

In experiments of biological activity the studied compounds were used at 2 mM concentration that showed less cytotoxicity than control agent (Fig. 2).

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Figure 2. The cytotoxicity of the compound 3 against human BJ fibroblasts (ATCC, USA).

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Compound 3 was used at the concentration ranges (16 – 0.125 mM). Control 1 represents the effect of 1.5 % ethanol solution used as compound 3 diluent (i.e. negative control). Cytotoxic

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agent etoposide (Calbiochem, USA) was used at 40 M as a positive control (control 2).

Compounds 2-4 showed anti-aggregation ability on the plasma status of IHD patients with

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pronounced alterations in the hemostasis system: the rate of the micro-platelet spontaneous aggregation decreased significantly and returned to the normal level in the presence of

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compounds 2-4. At the same time, compounds 2, 3 also reduced the activation of coagulation plasma factors equally (p < 0.05): the APTT normalized, the prothrombin time increased, and the addition of compound 4 to the plasma did not significantly affect these indicators. Moreover, all the tested substances did not affect the enzymatic activity of thrombin (Table 2). Table 2. The effect of compounds 2-4 on platelet aggregation and indicators of coagulation hemostasis in vitro Plasma

Aggregation rate,

N=8

rel.units/s

APTT, s

Prothrombin

Thrombin

time, s

time, s

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Journal Pre-proof < 0.05

23-36

11-15

14-21

Healthy donors

0.04±0.02

32±2.0

12.0±0.5

15.0±0.1

Patients with IHD

0.94±0.3

26.0±1.2

8.9±0.6

15.2±0.9

Compound 2

0.07±0.01*

29.3±2.5*

10.9±0.5*

15.1±0.2

Compound 3

0.05±0.02*

30.9±1.6*

9.8±0.4*

14.9±0.4

Compound 4

0.2±0.1*

28.0±1.4

9.2±0.1

15.0±0.6

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Normal values

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test results compared to samples for patients with IHD

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Note: N - number of measurements; * p < 0.05 - statistically significant differences of blood

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Spontaneous platelet aggregation rate was induced by the activation of specific receptors under the influence of various physiological or pathological factors (collagen, adrenaline, ADP, etc.).

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Our results showed that the studied compounds, in comparison with acetylsalicylic acid, showed a more significant inhibitory ability: they almost completely suppressed cell-induced platelet

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aggregation by epinephrine, ADP, collagen, immunocytophyte and reduced the effect of spontin

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(Table 3).

Table 3. Effect of compounds 2-4 on the in vitro induced platelets activation compared to

Inductor

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acetylsalicylic acid

Control,

Comp. 2,

Comp. 3,

Comp. 4, Acetylsalicylic

%

%

%

%

ADP

66±4

7±4 *

17.0±5.6* 14±4*

46.3 ± 9.1*

Adrenalin

67±6

0*

4±3*

20±5*

72±2

Arachidonic

68±4

4±4*

6±4*

18±3*

62±5

acid, %

acid

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Journal Pre-proof Collagen

72±9

8±6*

0*

0*

70±2

Ristocetin

70±6

20 ± 9*

27± 6*

38±6*

72±4

Note. Number of measurements: N=5; * p-authenticity ˂ 0.001 in comparison to results without substances.

The effects of compounds 2, 3 on thrombodynamic had also indicated inhibition of coagulation factors’ activation: the fibrin clot growth rate decreased (v - the growth inhibition rate, vi- the

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initial fibrin clot growth velocity, vst - the state velocity), density (D), fibrin clot size (СS) and

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spontaneous fibrin clots formation rate (Fsp) decreased (Table 4, Fig. 3).

Patients

min

min

39.4±4.1

vst,

CS,

D,

Fsp,

μm / min

μm / min

μm

rel. units

min

56.4±0.7

39.4±4.1

1390±35

31871±293

26.5±0.7

vi,

1.8±0.1

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with IHD

/ Tlag,

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Plasma

μm

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v,

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Thrombodynamic values

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Table 4. Thrombodynamic test results in the presence of compounds 2, 3

Comp. 2

23.5±1.2*

1.5±0.15* 42.4±2.8* 23.5±1.2* 979±45*

22505±344*

---

Comp. 3

24.1±1.4*

1.6±0.12* 43.2±0.8* 28.0±0.9

898±34*

27878±478*

---

Normal

20.5-30

0.8-1.5

833-

14000-

---

1173

32000

plasma

39.1-54.6

20.5-30

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Note: number of studies N=4; * - p < 0.05 - representing statistically significant differences of

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blood test results compared to plasma of patients with IHD.

Figure 3. Top line – plasma obtained from IHD patients on the 5th, 15th and 30th minutes of

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photo registration of the coagulation hemostasis activation without adding compounds; lower

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line - plasma from IHD patients in the presence of compound 3. Activation of platelets leads to their adhesion, aggregation and activation of plasma

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coagulation factors. These processes are accompanied by the platelets phospholipid surface transformation leading to the secretion of additional thrombogenic particles – microvesicles –

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into the bloodstream. The results of flow cytometry indicated the appearance of such particles and their quantity change in the platelet rich plasma. The number of platelets decreased during

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storage time, and the number of microvesicles increased significantly on the first day of storage, and then gradually decreased. In the presence of compounds 3 and 4 these changes were less significant (Table 5). Therefore, besides the inhibition of receptors activity of platelets, the tested substances also stabilized their cell membranes. Table 5. The results of flow cytometry of platelet concentrate The number of particlies in 1 μl (n =3) Incu-

Control

Compound 3

Compound 4

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Journal Pre-proof platelets

microvesicles platelets

98900

1h

59±9

24 h

2555±400

48 h

992±81

72 h

56±6

±120 96700 ± 180 96358 ±90 95636

99289±225*

747±150*

97867±350*

141±44*

97668±240*

19±5*

96911±225*

13±6*

99641±410*

452±98*

98267±356*

240±52*

97613±290*

5±3*

96773±650*

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±156

25±4*

microvesicles platelets

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time

microvesicles

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bation

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Note: N=3; * - p < 0.05 - statistically significant differences compared with control without

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compounds.

The results of standard coagulation tests, flow cytometry and thrombodynamics have revealed that compound 3, in contrast to compound 4, reduced the number of microvesicles in samples of

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thromboconcentrate, as well as almost completely suppressed their thrombogenic properties – all indicators normalized. Similar results were observed after the removal of microvesicles from the

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plasma of IHD patients (Fig. 4).

Figure 4. A - plasma of IHD patients after 30 minutes of photo registration of coagulation hemostasis activation; B - plasma of IHD patients in the presence of compound 3; C - plasma of sufferer with coronary artery disease after removal of microvesicles.

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Journal Pre-proof The ability of compounds 2 and 3 to suppress the thrombogenic properties of the cell membrane surface was confirmed by the results of experiments obtained with model DPC micelles. The addition of DPC micelles to the plasma showed their coagulation activity (the formation time of the fibrin clot in the APTT test was 71±4 s), which decreased in the presence of compound 3 (84±6 s). The interaction of the drug molecules with phospholipid membranes can be effectively studied by nuclear Overhauser effect (NOE) NMR spectroscopy40. However, this method has some constraints when applied in solution because of very short 1H spin-spin relaxation times leading

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to extensive broadening of the NMR signals. The solution of this problem can be found using

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model systems, such as dodecylphosphocholine (DPC) micelles. DPC has the same zwitterionic head group as phosphatidylcholine and can be used as a simple model for eukaryotic membranes.

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At the same time, DPC micelles are much smaller than phospholipid bilayers and, therefore, more suitable for NMR spectroscopy in solution as it was shown in works41–45.

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To study the effect of different substituents at the C-2 position on the interaction of bornane

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derivatives with phospholipid membranes, NMR experiments both in the presence and in the absence of DPC micelles (solution-state NMR for compounds 2-4) and POPC vesicles (solidstate NMR for compounds 3 and 4) were carried out.

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The proton NMR spectra of compounds 4 and 3 in D2O, DPC/D2O solutions are shown in Figs. 5 and 6, respectively. To study the spatial structure of the molecular complex of

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compounds 3 and 4 with DPC micelles, the 1H-1H two-dimensional NOESY experiments were

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carried out. However, only cross peaks corresponding to intramolecular NOEs were observed. As it can be seen from Fig. 6, the 1H NMR spectrum of compound 3 in D2O slightly differs from the spectrum in D2O in the presence of DPC. All signals of compound 3 are shifted to higher fields (Δδ up to -0.09 ppm) but the shape of the signals almost did not change in the presence of DPC micelles. The NMR lines remained well-resolved and no significant broadening was observed. In order to check if compound 3 intercalates the membrane mimetic, 2D DOSY experiments were carried out (Fig. 7). Significantly different diffusion coefficients were observed for DPC micelles (D = 8.00·10-11 m2/s) and for compound 3 (D = 1.63·10-10 m2/s). Furthermore, 2D DOSY NMR experiment showed that compound 4 and DPC micelles had rather different selfdiffusion coefficients in D2O solution (3.41·10-10 and 8.67·10-11 m2/s, respectively). These facts

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Journal Pre-proof indicate that compounds 3 and 4 do not bind to DPC micelles. Thus, no confirmation of stable complex formation between DPC micelles and compounds 3 and 4 were obtained from solution-

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state NMR data.

Figure 5. The proton NMR spectra of compound 4 in D2O (a) and in DPC/D2O solutions (b) at temperature 30°C. Numbers designates protons of compound 4, letters A-H belong to DPC protons. The structure of the DPC molecule with assignment of proton groups is shown at the top of spectra.

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Figure 6. The proton NMR spectra of compound 3 in in D2O (a) and in DPC/D2O solutions (b) at temperature 30°C. Numbers designates protons of compound 3, letters A-H belong to DPC

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of spectra.

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protons. The structure of the DPC molecule with assignment of proton groups is shown at the top

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Figure 7. The 2D DOSY NMR spectrum of compound 3 in DPC/D2O solution at temperature

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30°C.

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There was high probability of interaction of compound 2 with DPC micelles, because of its poorly solubility in water. Compound 2 was initially studied in CDCl3 solution (Fig. 8a). The proton signals were assigned using correlations in 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC NMR experiments (see Supplementary data) and thus the chemical structure of compound 2 after synthesis was definitely confirmed. However, compound 2 was found to be soluble in D2O in the presence of DPC micelles. Proton NMR spectrum of the compound 2 in CDCl3 solution significantly differs from those in DPC/D2O solution (Fig. 8). All signals were broadened and shifted to higher fields. Such changes in the NMR spectrum can be caused by interactions of compound 2 with DPC micelles and possible formation of complex between them.

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Figure 8. The proton NMR spectra of compound 2 in CDCl3 (a) and DPC/D2O solutions (b) at

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temperature 30°C. Numbers designates protons of compound 2, letters A-H belong to DPC of spectra.

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protons. The structure of the DPC molecule with assignment of proton groups is shown at the top

To verify if compound 2 is bonded to DPC micelles and to study the structural details of intermolecular complex, 2D NOESY NMR experiment were carried out (Fig.9). Unfortunately, most of possible intermolecular cross peaks were overlapped with those arising from intramolecular NOEs. Nevertheless, clearly distinguishable cross peaks between the proton H-2 of compound 2 and the H, F and E protons of DPC were observed in the spectrum. Therefore, it was concluded that compound 2 formed a molecular complex with DPC micelles and the compound penetrated relatively deeply into the hydrocarbon core by its cyclic part. Such behavior also was observed in recently published works46–50. The location and orientation of the

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Journal Pre-proof substituent –SCH2COOH could not be determined from the NMR data because of the fact that the only one 1H NMR signal CH2-11 of this fragment were overlapped with the D signal of DPC. The additional 2D DOSY NMR experiment also confirm the fact that compound 2 formes a

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molecular complex with DPC micelles (see Fig. 10S in Supplementary material).

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Figure 9. The fragment of 2D NOESY NMR spectrum of compound 2 in DPC solution at temperature 30°C. Numbers correspond to the atomic groups in compound 2, letters D-H refer to the hydrocarbon groups of DPC. Intermolecular crosspeaks are highlighted by grey circles.

Compounds 3 and 4 were studied in the presence of POPC membranes using phosphorus (31P) and deuterium (2H) NMR experiments. Firstly, we performed solid-state phosphorus NMR experiments in order to verify the biologically relevant liquid-crystalline phase state of the phospholipid membranes in the presence of the terpenoids. Figure 10 shows

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P and 2H NMR

spectra of D-POPC in the absence and in the presence of compounds 3 and 4 at a molar ratio of 4:1. As shown in Fig 10, the typical

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P NMR line shape of a lamellar liquid crystalline bilayer

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Journal Pre-proof remains in the presence of 20 mol% of these compounds. The chemical shift anisotropy values, Δσ were determined by simulations of the line shape of solid-state

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P NMR spectra lines (red

lines). In the presence of compound 4, the value of Δσ insignificantly drops from 45.5 ppm (for pure POPC membranes) to 44.0 ppm, whereas in the presence of compound 3 it decreases more significantly to 41.0 ppm. Such changes in the

31

P chemical shift anisotropy values can be

explained by a higher of molecular mobility or changes in the orientation of the POPC head

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group in the presence of studied compounds.

Figure 10. The 31P (left column) and 2H (right column) NMR spectra of D-POPC in the absence (A and D) and in the presence of compounds 3 (B and E) and 4 (C and F) at 50 wt % H2O at 30°C. The mole fraction of small molecules of compounds (3 and 4) and POPC was 1:4. The red lines shows numerically obtained simulated shape for the wide line static 31P solid-state spectra with a chemical shift anisotropy of Δσ = 45.5 ppm (A), Δσ = 41.0 ppm (B), and Δσ = 44.0 ppm (C).

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Figure 11. Order parameter profiles of the 2H NMR spectra at temperature 30 °C. The order parameter profiles are shown for D-POPC in the presence of the compounds 3 (▲, red triangles),

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4 (▼,green triangles) and in the absence (□, blue squares).

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To study of the influence of the two compounds on the hydrocarbon core of the lipid membrane, H NMR measurements were performed and chain order parameters (shown in Fig. 11) were

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calculated. Compared to a pure POPC-d31 membrane, the chain order parameter in the presence of compound 4 are very similar, only a small decrease in middle/lower chain region is visible. In

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contrast to this, addition of compound 3 drastically decreased the chain order parameters of the POPC membrane over the whole chain region which points to a significant increase in the amplitude of motion along the acyl chains. This is also reflected in the calculated lipid chain length Lc* according the mean torque model35, which amounts to 11.5 Å for pure POPC-d31 and remained nearly the same in the presence of compound 4 but drastically decreased to 10.5 Å in the presence of compound 3. To obtain further insights in the membrane location of the studied compounds, 1H NOESY MAS NMR measurements were carried out. Due to the high mobility and molecular disorder in lipid membranes in the physiologically relevant liquid-crystalline phase, the obtained crossrelaxation rates reflect a contact probability between the respective molecular groups. Therefore, the quantitative analysis of the cross-relaxation rates between the compound and the different

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Journal Pre-proof molecular segments of POPC can be used to obtain a distribution function of the studied molecule in the lipid membrane, as it was shown for some small molecules39,51–54. The obtained cross-relaxation rates between the cyclic fragments of compounds 3 and 4 and various protons of POPC, are shown in Figs. 12 and 13. A direct comparison of the absolute values of the cross relaxation rates for compound 3 and 4 is difficult due to the influence of the correlation time of motion on the cross-relaxation rates. Both molecules exhibit a quite vast distribution function along the perpendicular to the membrane’s plane reflecting a high degree of mobility of these

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molecules inside the lipid membrane.

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Figure 12. The cross-relaxation rates of the highlighted molecular group of compound 4 to the molecular fragmentsof POPC in planar membranes. * means that these cross-peaks were not analyzed due to possible signal overlap with CH3 groups signal of compound 4 and POPC.

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Journal Pre-proof Figure 13. The cross-relaxation rates of the highlighted molecular groups of compound 3 to the molecular segments of POPC.* means that these cross-peaks were not analyzed due to possible signal overlap with signal of compound 3 and POPC.

While for compound 4 the maximum of distribution function for the CH3 group at the gem dimethyl fragment is clearly found deep inside the hydrocarbon core of the membrane, for compound 3 this distribution profile is clearly shifted to the middle and upper chain area. This shift is a result of the action of more polar carboxyl group of the molecule, which physical

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interactions with the phospholipid molecules forces the molecules to move in the direction of the

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head group of POPC.

Moreover, for compound 3 the second distribution profile for the CH2-group at the thio

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methylene fragment was determined, which as it turned out is located even more close to the

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upper section of the chain. Therefore, the studied compound 3 is located predominantly in the

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chain segments, oriented by polar side to the direction of the lipid-water surface. Conclusion

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The results from this study suggest that among all studied terpenoids, salt (compound 3) may be the most promising for further research because of its better effects on spontaneous

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aggregation compared with isoborneol (compound 4), and due to its higher solubility in water compared with sulfide (compound 2), which has approximately the same antiaggregatory and

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anticoagulant properties. The findings of this study indicate that salt (compound 3), has a membrane location, which is shifted somewhat in the direction of the membrane-water surface, which shields the hydrophobic interactions of coagulation factors from the surface of the membrane. Apparently, the absence of such properties in isoborneol (compound 4) is due to its large immersion inside the membrane. In our view, these results are an excellent initial step toward creating efficient drugs based on the newly synthesized thioterpenoids targetted to increase the effectiveness of prevention and treatment of cardiovascular diseases, the prevention of the development of acute vascular thrombosis and many other diseases accompanying disorders in the human hemostasis system.

29

Journal Pre-proof ACKNOWLEDGMENTS This paper is dedicated to the late Dr.associate professor Leisan F. Galiullina, the scientist, who made a significant contributionto this research, and our colleague, whose memory will remain forever in our hearts. The work was sponsored by a subsidy under the auspices of state support to KFU for improving its competitiveness among leading global science and education centers. V.V. Klochkov acknowledges the state assignment in the sphere of scientific activities (project

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no. 3.5283.2017/6.7). H.A. Scheidt acknowledges the support of Deutsche Forschungsgemeinschaft (DFG, German research foundation) project SCHE 1755/4-1.The NMR study was

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carried out with financial support of Ministry of Science and Higher Education and of the

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Russian Federation (projects 01201260481 and 01200950825), and under grant of Council on

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grants of the President of the Russian Federation (project MK-1409.2019.3).

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* Correspondences:

Scheidt Holger A. Institute for Medical Physics and Biophysics, Leipzig University, Härtelstr.

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16–18, Leipzig, 04107, Germany [email protected]

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Nikitina, Liliya E. Department of General and Organic Chemistry, Kazan State Medical

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University, Kazan, Russian Federation [email protected] Khodov, Ilya A. Institute of Physics, Kazan Federal University, Kazan, 420008, Russian Federation, G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, 1 Akademicheskaya Street, Ivanovo, 153045, Russian Federation [email protected]

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Journal Pre-proof Author Contribution Statement

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Liliya E. Nikitina: Writing- Original draft preparation., Roman S. Pavelyev: Synthesis of new compounds, Valeriya A. Startseva: Sample preparation., Sergei V. Kiselev: Investigation of biochemical assays, Leisan F. Galiullina: The solution-state NMR interpretation, Oksana V. Aganova: The model membrane sample preparation., Ayzira F. Timerova: Soultion-state NMR experiment, Sergei V. Boichuk Cytotoxicity experimet interpretation, Zulfiya R. Azizova Cytotoxicity experimet, Vladimir V. Klochkov: Methodology of the solution state experiment, Daniel Huster: Solid-state NMR data curation, Editing, Ilya A. Khodov: Conceptualization, Solid-state NMR experiment, Writing- Reviewing and Editing, Holger A. Scheidt: Methodology of solid-state NMR, Validation and Editing.

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof Highlights A novel thioterpenoids with good coagulation activity was synthesized. The antiaggregatory and anticoagulant properties were examined. The molecular interaction of thioterpenoids with phospholipid membranes has been explained by various reliable NMR methods. The location of molecule of novel triterpenoid into membrane is shifted somewhat in the direction of the membrane-water interface

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This paper has potent application in the area of chemical, pharmaceutical and medical science.

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