Sperm motility plays an important role in normal fertilization process of mammalian. Successful in vitro-oocyte fertilization by artificial insemination technique requires sufficient sperm motility (Yanagimachi, 1966). Role of external factors on regulation of sperm motility has been studied extensively. Ionized calcium ion stimulates sperm motility in some studies (Fakih et al., 1986; Lornage et al., 1983), meanwhile lack and/or inhibition of activity have been observed and reported by others (Hong et al., 1985; Umeyama et al., 1986). Versatility of mechanisms involved and/or multiple intracellular sites activity of calcium ions, as well as variation in experimental design could be mentioned as the underlying cause of controversial data and results observed. In order to elucidate the role of ionized calcium in regulation of human sperm motility, an in vitro study was conducted to evaluate the activity of exogenous calcium ion, EDTA and diltiazem on mean velocity and percent motility of human sperms (Aaberg et al., 1989). The results indicated that a minimum level of extracellular ionized calcium is essential for normal sperm motility and also suggests that regulation of sperm motility are more dependent on the intracellular translocation of calcium ions than the extracellular ionized calcium concentration. A reversible male infertility episode after administration of Calcium Channel Blocker (CCB) to control hypertension has been reported to be responsible for failures in an IVF trial case (Benoff et al., 1994). Insertion of lipophilic calcium ion antagonists into the lipid bilayer of the sperm plasma membrane has been proposed as a potential mechanism for inhibition of sperm fertilization.
The possible role of L-type Ca2+ channels on the acrosomal reaction of human spermatozoa has also been investigated. The results demonstrated that acrosomal reaction of human spermatozoa is highly associated with L-type Ca2+ channels and is mainly mediated by calcium influx through α-1H-T-type Ca2+ channels (Weon-Young et al., 2000). Diltiazem and Nifedipine are both an L-type calcium channel blocker, that when used, the first one cause a drastic and irreversible inhibitory activity of sperm motility in vitro along with significant reduction of sperm viability and the second demonstrated acrosomal inhibitory activity (Wood et al., 2003). The efficacy of dihydropyridine compounds as inhibitors of acrosomal reaction and their potential contraceptive activity has been investigated by (Kirkman-Brown et al., 2003). The subsequent studies revealed acrosomal reaction is the result of progesterone induction and consequently, Nifedipine inhibits progesterone-induced acrosomal reaction in human sperm. In the present study, the activity of seven newly synthesized dihydropyridine analogues on the motility of sperm were determined and compared to nifedipine activity that was used as standard.
MATERIALS AND METHODS
The candidate compounds were synthesized and evaluated in the department of Medicinal Chemistry and physiology faculties of pharmacy and Medicine respectively, during Dec. 2005- Apr. 2007.
Melting points were determined using a kofler hot stage apparatus and are uncorrected. 1HNMR spectra were obtained on a Bruker FT-80 spectrometer (Bruker, Rheinstetten, Germany). TMS was used as internal standard. Mass spectra were measured with a Finnigan TSQ-70 spectrometer (Finnigan Mat, Breer, Germany) at 70 ev. Infrared spectra were acquired on a Nicolet 550-FT spectrometer (Madison, WI, USA).
Ham's F-10 medium were obtained from Sigma-Aldrich Chemie Gmbh (Deisenhofen, Germany).
Measurement of spermatozoa motility was determined according to the published methods (Gardner et al., 2006; Placzek et al., 1987).
Chemistry: Methyl, ethyl, i-propyl, i-butyl, t-butyl and benzyl acetoacetate were purchased from Sigma-Aldrich Chemie Gmbh (Deisenhofen, Germany). Cyclohexyl acetoacetate and 2-Chlorothiobenzamide (2) were prepared according to the recommended methods from cyclohexanol (Clemens and Hyatt, 1985) and 2-chlorobenzonitrile (Fairfull et al., 1952) and the corresponding reagents respectively (Scheme 1).
Preparation of 4-chloromethyl-2 (2-chlorophenyl) thiazole (3): To a stirred solution of 1, 3-dichloroacetone (0.1 mole) in toluene (100 mL) was added 2-chlorothiobenzamide (2) (0.1 mole) and refluxed at 110°C for 3 h. The reaction was monitored by TLC. The solvent was removed under reduced pressure. To the residue, water (200 mL) was added and the precipitated material was filtered. The crystallization was carried out from methanol to give 3. m.p. 61-63°C, % yield: 68. 1H NMR (CDCl3): δ 4.78 (s, 2H, CH2), 7.20 (s, 1H, H5), 7.37-7.8 (m, 4H, phenyl and H5 thiazole).
Preparation of 4-hydroxymethyl-2- (2-chlorophenyl) thiazole (4): To the compound 3 (10 g) was added a mixture solution of sulfuric acid (50%) and refluxed for 24 h. After cooling, the pH was neutralized with concentrated solution of sodium hydroxide, the precipitation was filtered and recrystallized from chloroform and petroleum ether. m.p. 87-88°C, % yield: 83. 1H NMR (CDCl3): δ 4.88 (s, 2H, CH2), 5.40 (bs, 1H, OH), 7.06-7.90 (m, 5H, phenyl and H5 thiazole). IR (KBr): v 3300 (OH) cm-1.
Preparation of 2- (2-chlorophenyl) thiazol-4-carboxaldehyde (5): To compound 4 (2 g) was added MnO2 (6 g) in dichloro methane (50 mL) and stirred over night. The mixture was filtered on diatomaceous earth and the solvent removed under reduced pressure. The residue was crystallized from MeOH: H2O to give aldehyde of 5. m.p. 58-60°C, yield: 70. 1H NMR (CDCl3): δ 7.2-7.7 (m, 3H), 8.15-8.36 (m, 2H), 10.14 (s, 1H, CHO), IR (KBr): v 1697 (C = O) cm-1.
Preparation of dimethyl 1,4-dihydro-2,6-dimethyl-4- (2- (2-chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6a): To a solution of ammonium acetate (1 g) in methanol (20 mL) was added 5 (2 mmol) and methyl acetoacetate (4 mmol). The mixture was refluxed for 5 h. The solvent was removed under reduced pressure. To the residue was added water (10 mL) and extracted with ethyl acetate (2x30 mL), dried by sodium sulphate and removed under reduced pressure. The residue was crystallized in MeOH: H2O to give compound 6. m.p. 194-195°C, yield: 48. 1H NMR (CDCl3): δ 2.34 (s, 6H, CH3), 3.72 (s, 6H, OCH3), 5.34 (s, 1H, H4), 5.88 (bs, 1H, NH), 7.01 (s, 1H, H5-thiazole), 7.20-7.50 (m, 3H, aromatic), 8.01-8.02 (m, 1H, aromatic), IR (KBr): v 3329 (NH), 1700 cm-1 (C = O), MS m/z (%) 419 (100), 359 (30), 355 (7), 224 (30), 149 (8), 131 (30), 116 (22), 71 (14), 59 (49).
Preparation of diethyl 1,4-dihydro-2,6-dimethyl-4- (2- (2-chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6b): Compound of 6b was prepared similar to 6a, m.p. 143-145°C, yield: 48. 1H NMR (CDCl3): δ 1.24 (t, J = 7 Hz, 6H, CH3), 2.28 (s, 6H, CH3), 4.15 (q, J = 7 Hz, 4H, CH2), 5.32 (s. 1H, H4), 6.27 (bs, 1H, NH), 7.05 (s, 1H, H5-thiazole), 7.20-7.50 (m, 3H, aromatic), 8.01-8.15 (m, 1H, aromatic), IR (KBr): v 3338 (NH), 1695 (C = O), MS m/z (%) 447 (15), 373 (29), 359 (15), 278 (15), 252 (86), 224 (100), 196 (30), 150 (20), 77 (15), 57 (30).
Preparation of di-isopropyl 1,4-dihydro-2,6-dimethyl-4- (2- (2 chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6c): Compound of 6c was prepared similar to 6a, m.p. 145-146°C, yield: 47. 1H NMR (CDCl3): δ 1.03-1.29 (two d, J = 6.2 Hz, 12H, CH3), 2.29 (s, 6H, CH3), 4.95-5.11 (m, 2H, CH), 5.29 (s, 1H, H4), 5.92 (bs, 1H, NH), 7.04 (s, 1H, H5-thiazole), 7.37 (m, 3H, aromatic), 8.06-8.22 (m, 1H, aromatic), IR (KBr): v 3354 (NH), 1694 (C = O), MS m/z (%) 475 (8), 446 (28), 386 (15), 373 (45), 345 (28), 280 (21), 251 (94), 195 (100), 148 (42), 105 (21), 56 (37).
Preparation of di-isobuthyl 1,4-dihydro-2,6-dimethyl-4- (2- (2-chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6d): Compound of 6d was prepared similar to 6a, m.p. 101-102°C, yield: 33. 1H NMR (CDCl3): δ 0.91 (d, J = 6.6 Hz, 12H, CH3), 1.93 (m, 2H, CH (CH3) 2), 2.2 (s, 6H, CH3), 3.89 (d, J = 6.2 Hz, 4H, OCH2), 5.38 (s, 1H, H4), 6.23 (bs, 1H, NH), 7.05 (s, 1H, H5-thiazole), 7.32 (m, 3H, aromatic), 8.1 (m, 1H, aromatic), IR (KBr): v 3339 (NH), 1690 (C = O), MS m/z (%) 503 (9), 501 (15), 427 (15), 400 (22), 343 (15), 307 (36), 251 (15), 194 (43), 148 (15), 105 (5), 66 (15), 56 (100).
Preparation of di-tertiarybutyl 1,4-dihydro-2,6-dimethyl-4- (2- (2-chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6e): Compound of 6e was prepared similar to 6a, m.p. 163-165°C, yield: 22. 1H NMR (CDCl3): δ 1.45 (s, 18H, t-Bu), 2.28 (s, 6H, CH3), 5.32 (s, 1H, H4), 5.75 (bs, 1H, NH), 7.09 (s, 1H, H5-thiazole), 7.25 (m, 3H, aromatic), 8.01-8.15 (m, 1H, aromatic), IR (KBr) v: 3338 (NH), 1691 (C = O), MS m/z (%) 503 (9), 501 (20), 430 (8), 402 (15), 399 (43), 345 (15), 306 (87), 251 (21), 194 (100), 150 (21), 128 (14), 56 (87).
Preparation of di-benzyl 1,4-dihydro-2,6-dimethyl-4- (2- (2- chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6f): Compound of 6f was prepared similar to 6a, m.p. 117-118°C, yield: 28. 1H NMR (CDCl3): δ 2.33 (s, 6H, CH3), 5.15 (m, 4H, CH2), 5.35 (s, 1H, H4), 5.96 (s, 1H, NH), 6.66 (bs, 1H, H5-thiazole), 7.05-7.45 (m, 13H, aromatic), 8.02-8.22 (m, 1H, aromatic), IR (KBr): v 3308 (NH), 1695 (C = O), MS m/z (%) 571 (15), 503 (58), 435 (29), 427 (86), 401 (94), 327 (37), 307 (100), 253 (29), 196 (65), 195 (100), 150 (29), 91.7 (58).
Preparation of di-cyclohexyl 1,4-dihydro-2,6-dimethyl-4- (2- (2-chlorophenyl) thiazol-4-yl) -3,5-pyridine dicarboxylate (6g): Compound of 6g was prepared similar to 6a, m.p. 149-151°C, yield: 33. 1H NMR (CDCl3): δ 1.07 (m, 20H, cyclohexyl), 2.28 (s, 6H, CH3), 4.83 (m, 2H, CO2CH-cyclohexyl), 5.32 (s, 1H, H4), 6.27 (bs, 1H, NH), 7.05 (s, 1H, H5-thiazole), 7.20-7.50 (m, 3H, aromatic), 9.01-8.13 (m, 1H, aromatic), IR (KBr): v 3325 (NH), 1691 (C = O), MS m/z (%) 555 (14), 502 (21), 435 (29), 401 (43), 376 (59), 308 (64), 300 (21), 252 (14), 196 (100), 150 (29), 90 (100).
Semen samples and sperm preparation: An andrological outpatient clinic
supplied the required semen samples (H. R. Sadeghipour Roodsari, Noor Laboratory
Keshavarz Blvd Karegar. Ten healthy donors with informed consent after 48-72
h of abstinence supplied the required semen samples via masturbation that were
collected into plastic containers. The samples complied with WHO required sample
specification and guideline and processed immediately upon liquefaction. After
liquefaction at 37°C for 30 min, the spermatozoa were separated from seminal
plasma by two consecutive washing (300 g, 5 min) with PBS (phosphate-buffer
saline, pH 7.4: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4,
8.1 mM Na2HPO4 in distilled water). The semen samples
were allowed to swim-up for 1 h at 37°C in 1 mL of Ham's F-10 medium containing
10% HSA (human serum albumin) and subsequently the supernatant were carefully
aspirated. The sperm concentration was adjusted to 1 million per millilitre
for subsequent steps. Nifedipine, a calcium channel blocker and seven synthesized
dihydropyridine compounds (6a-g) were dissolved in DMSO that were used as solvent
medium. To evaluate the activity of compounds, 95 μL of motile sperm samples
(1 million per mL) were incubated with 5 μL of synthesized compounds (6a-g)
(1x10-2 M) to obtain the final concentrations of 5x10-4
M in volume of 100 μL. Nifedipine and DMSO were used as standard and blank
respectively. All incubations were performed at 37°C in presence of 5% CO2.
Sperm motility were measured during incubation process after 1 and 2 h of contact
Human sperm motility studies: The percentage of motile sperms were assessed by visual inspection of 100 sperms. The degree of sperm movement for every encountered sperm was graded as a, b, c or d and defined as follows: a: rapid progressive motility; b: slow progressive motility; c: non-progressive motility and d: immotility (WHO, 1999). Total motility (a + b + c) was reported. All samples used in this study had values of immotile spermatozoa (grade d) lower than 20% of the total value. The count of spermatozoa were carried out by x400 multiplied microscope (Zeiss).
Statistical analysis: Data was analysed using a computer software pakage (SPSS ver 11.5 Chicago, Ill.). Paried t-Test statistical analysis was provided within the pakage. The data was shown as mean±SD and the p-value <0.5 considered as significant.
RESULTS AND DISCUSSION
Chemistry: Treatment of 2-chlorothiobenzamide (2) and 1,3-dichloroacetone
render compound (3). That when refluxed in acidic media led to the 2 hydroxy
methyl derivatives of thiazol (4). Oxidation of hydroxymethyl group by MnO2
in dichloromethane resulted aldehyde (5). The compounds (6a-g) were prepared
according to the conventional procedures for synthesis of dihydropyridine compounds
||Reagents: (a) SH2, TEA/pyridine, (b) 1,3-dichloroacetone,
(c) H2SO4/H2O, (d) MnO2/CH2Cl2
and (e) CH3COCH2COOR, ammonium acetate/MeOH
In vitro study: Nifedipine were mentioned previously as the agent responsible for failure of an IVF trial case due to sperm immobilizing activity (Benoff et al., 1994). In this study, the sperm motility activity inhibition of nifedipine were used as a reference to evaluate the potential motility activity inhibition of seven candidate compounds. Table 1 shows the results obtained after determination of total sperm motility in the presence of the seven compounds and Nifedipine after 1 and 2 h of incubation period.
Nifedipine reduced motility completely both after 1 and 2 h of incubation. Sperm motility reduced value for candidate compounds (6a-g) shows a gradual increase proportional to the size elongation of alkyl ester group. As a result the methyl ester of the test compounds with 50% of activity in comparison to nifedipine (in 2 h group) is the most active compound (p<0.05). Though the other members of the synthetic compounds did not statistically show a significant activity differences in comparison to DMSO. Therefore, the results could be attributed to the point that reducing the size of ester group (R1) is critical in the activity of the compounds. Increase in the size from methyl to other lipophilic compounds led to the reduction of the activity.
This research was supported by grants from INSF (Iran National Science Foundation) and the Research Council of Tehran University of Medical Sciences.