1-[(Imidazolidin-2-yl)imino]-1H-indoles as new hypotensive agents: synthesis and in vitro and in vivo biological studies

Article abstract of DOI:10.1111/cbdd.12846

A series of 1-[(imidazolidin-2-yl)imino]-1H-indole analogues of hypotensive α₂-AR agonists, 1-[(imidazolidin-2-yl)imino]-1H-indazoles, was synthesized and tested in vitro for their activities at α₁- and α₂-adrenoceptors as well as imidazoline I₁ and I₂ receptors. The most active 1-[(imidazolidin-2-yl)imino]-1H-indoles displayed high or moderate affinities for α₁- and α₂-adrenoceptors and substantial selectivity for α₂-adrenoceptors over imidazoline-I₁ binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that substitution at C-7 position of the indole ring may result in compounds with high cardiovascular activity. Among them, 7-fluoro congener 3g showed the most pronounced hypotensive and bradycardic activities in this experiment at a dose as low as 10?μg/kg i.v. Metabolic stability of the selected compounds of type 3 was determined using both in vitro and in silico approaches. The results indicated that these compounds are not vulnerable to rapid first-phase oxidative metabolism.

Full text of DOI:10.1111/cbdd.12846

Received Date : 31-May-2016
Revised Date : 13-Jul-2016
Accepted Date : 19-Aug-2016
Article type : Research Article
1-[(Imidazolidin-2-yl)imino]-1H-indoles as New Hypotensive Agents: Synthesis, In Vitro
and In Vivo Biological Studies
Anita Kornickaa,*, Aleksandra Wasilewskaa, Jarosław Sączewskib, Alan L. Hudsonc,
Konrad Boblewskid, Artur Lehmannd, Karol Gzellaa, Mariusz Belkae, Franciszek
Sączewskia, Maria Gdaniecf, Apolonia Rybczyńskad, Tomasz Bączeke
aDepartment of Chemical Technology of Drugs, Medical University of Gdańsk,
80-416 Gdańsk, Poland
bDepartment of Organic Chemistry, Medical University of Gdańsk,
80-416 Gdańsk, Poland
cDepartment of Pharmacology, 947 Medical Sciences Building, University of Alberta,
Edmonton T6G 2H7, Canada
dDepartment of Pathophysiology, Medical University of Gdańsk, 80-416 Gdańsk, Poland
eDepartment of Pharmaceutical Chemistry, Medical University of Gdańsk, 80-416 Gdańsk,
Poland
fFaculty of Chemistry, A. Mickiewicz University, 60-780 Poznań, Poland
a
*Corresponding author: Anita Kornicka, Department of Chemical Technology of Drugs,
Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland
e-mail: a.kornicka@gumed.edu.pl, tel.: +48-58-349-19-56; fax: +48-58-349-16-54
Abstract:
A series of 1-[(imidazolidin-2-yl)imino]-1H-indole analogues of hypotensive α2-AR agonists
1-[(imidazolidin-2-yl)imino]-1H-indazoles, was synthesized and tested in vitro for their
activities at α1- and α2-adrenoceptors as well as imidazoline I1 and I2 receptors. The most
active 1-[(imidazolidin-2-yl)imino]-1H-indoles displayed high or moderate affinities for α1-
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doi: 10.1111/cbdd.12846
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and α2-adrenoceptors and substantial selectivity for α2-adrenoceptors over imidazoline-I1
binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that
substitution at C-7 position of the indole ring may result in compounds with high
cardiovascular activity. Among them, 7-fluoro congener 3g showed the most pronounced
hypotensive and bradycardic activities in this experiment at a dose as low as 10 μg/kg i.v.
Metabolic stability of the selected compounds of type 3 were determined using both in vitro
and in silico approaches. The results indicated that these compounds are not vulnerable to
rapid first phase oxidative metabolism.
Key words: imidazolines, indoles, alpha-adrenoceptor ligands, circulatory activities,
metabolic stability
Introduction
α-Adrenergic receptors (α-ARs) are involved in multiple physiological functions,
particularly cardiovascular regulation. For example, activation of central α2-ARs plays a
prominent role in lowering blood pressure and, thus, it has been utilized clinically for many
years in the treatment of hypertension (1,2). Other pharmacological effects of central α2-ARs
stimulation include behavioral functions, sedation, anxiolysis, analgesia and anaesthetic-
sparing activity (3,4). α2-ARs also mediate in insulin secretion, gastrointestinal motility, body
temperature as well as seizure threshold (3,5-6). On the other hand, stimulation of α1-ARs
may protect from abnormal heart rate (7), whereas α1-AR antagonists behave as vasodilators
(8). The blockade of α1-ARs may also be useful for the treatment of benign prostatic
hyperplasia (8,9).
It is well known that imidazoline-containing compounds, such as clonidine or
tolazoline, constitute the most vital set of drugs that interact with α-ARs (Figure 1, structure
A). In addition, imidazoline I1 binding sites (I1-IBS or I1-IRs for I1-imidazoline receptors)
have been postulated to participate in central antihypertensive effects of clonidine-like drugs
(10,11). Thus, rational development of novel imidazoline-containing drugs has proven to be
difficult owing to possible interaction with either α1-ARs and α2-ARs or I1-imidazoline
receptors. Moreover, it was found that even minor structural modification of the imidazoline-
containing compounds could significantly affect their receptor affinity and selectivity (12,13).
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Recently our SAR studies of imidazoline-containing α-adrenergic agents showed that
the isosteric replacement of the indazole ring in 1-[(imidazolin-2-yl)methyl]indazoles B by
the indole moiety led to compounds C (Figure 1), which exert different functional properties
with respect to α-ARs. Indeed, imidazoline-containing indazoles B exhibited hypotensive
properties due to α1-AR antagonist activity (14), while their indole analogues C were
described as partial α2A-AR agonists showing clonidine-like cardiovascular pattern (15).
Furthermore, we described 1-[(imidazolidin-2-yl)imino]-1H-indazoles D (Figure 1,
marsanidine-like ligands), which proved to be hypotensive α2-AR agonists (16-18). However,
pharmacological effects of the displacement of the indazole ring in compounds D with the
indole ring system have not been investigated previously. Therefore, the aim of this work was
to determine the influence of the replacement of the indazole ring in 1-[(imidazolidin-2-
yl)imino]-1H-indazoles D with the indole moiety on α-adrenergic affinity and selectivity as
well as cardiovascular effects of the indole analogues 3, thus obtained (Figure 1).
Methods and Materials
Chemistry
Melting points were measured on a Boetius apparatus and are uncorrected. IR spectra
were taken on a Perkin-Elmer FTIR 1600 spectrometer. NMR spectra were recorded on a
1
Varian Gemini 200 or a Varian Unity 500 apparatus. H and 13C chemical shifts were
measured relative to the residual solvent signal at 7.26 ppm and 77.0 ppm (CDCl3) or 2.50
ppm and 39.5 ppm (DMSO-d6). Preparative thin layer chromatography was performed on
silica gel 60 PF254 containing gypsum (Merck) with aid of Chromatotron® using the reported
solvent systems. 1-Amino-1H-indoles 2a (19), 2d (20) and 2e (21) were obtained according
to published methods by reacting the commercially available 1H-indoles 1a, 1d and 1e with
hydroxylamine-O-sulfonic acid (HOSA) as amination agent (20). 1-Amino-1H-indoles 2b
(22), 2c (23) and 2j (24) were synthesized as previously described from the commercially
available 1H-indoles 1b-c and 1j using monochloramine as amination agent (23). The same
procedure was applied to the synthesis of the previously not described 1-amino-1H-indoles
2f-i (Appendix S1, Supporting Information). N-tert-butoxycarbonyl-2-methylthio-4,5-
dihydro-1H-imidazole was obtained by reacting 2-methylthio-2-imidazoline with di-tert-
butyl dicarbonate (25). N,N′-bis(tert-butoxycarbonyl)imidazolidine-2-thione was synthesized
from imidazolidine-2-thione and di-tert-butyl dicarbonate (26).
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Preparation of 1-[(Imidazolidin-2-yl)imino]-1H-indoles 3a-j
Synthesis of 1-[(imidazolidin-2-yl)imino]-1H-indoles 3a-i. Compounds 3a-i were
obtained according to the method described by Sączewski et al. (27). A solution of the
appropriate 1-amino-1H-indole (2a-i) (4.6 mmol) and N-tert-butoxycarbonyl-2-methylthio-
4,5-dihydro-1H-imidazole (1.1 g, 5.1 mmol) in acetic acid (3 mL) was stirred at 62-65 °C (oil
bath) for 16 h. Then the solvent was evaporated under reduced pressure, and the oily residue
was treated with water (7 mL). The resulting suspension was made alkaline with 5% aqueous
NaOH solution to pH 9.5 – 10, and extracted with dichloromethane (3 x 10 mL). The
combined organic layers were dried over MgSO4 and concentrated under vacuum. The crude
product thus obtained was purified by preparative thin layer chromatography, followed by
crystallization from suitable solvent if necessary.
The free bases 3a-i were then converted into the corresponding water-soluble
hydrochlorides with use of ca. 1.6 M methanolic solution of hydrochloride (in the case of 3a,
3d-e), or ca. 2.5 M ethereal solution of hydrochloride (in the case of 3b-c, 3f-h) as well as
hydrobromide salt with use of 48% hydrobromic acid solution (in the case of 3i).
1-[(Imidazolidyn-2-yl)imino]-1H-indole (3a). Yield 0.50 g (55%); mp 140-142 °C
(ethyl acetate/methanol, 9.5:0.5 v/v); IR (KBr, cm−1) νmax 3396, 3203, 2883, 1634, 1605,
1505, 1453, 1324, 1286, 1219, 1075, 1042, 745, 716; 1H NMR (200 MHz, DMSO-d6) δ 3.23-
3.43 (m, 2H), 5.97 (s, 1H), 6.32 (d, J = 3.0 Hz, 1H), 6.57 (s, 1H), 6.89-7.07 (m, 2H), 7.13-
13
7.15 (m, 2H), 7.50 (d, J = 7.7 Hz, 1H); C NMR (50 MHz, DMSO-d6,) δ 42.2, 42.8, 97.7,
109.9, 118.3, 120.3, 120.6, 126.0, 127.9, 134.6, 166.0. Anal. Calcd for C11H12N4 (200.24): C,
65.98; H, 6.04; N, 27.98%. Found: C, 65.82; H, 5.97; N, 27.84%. Hydrochloride of 3a. Mp
211-213 °C (crystallization from ethanol/diethyl ether, 1:5 v/v); IR (KBr, cm−1) νmax 3256,
3154, 3090, 2893, 1667, 1630, 1457, 1309, 1225, 1081, 1048, 754. Anal. Calcd for
C11H13ClN4 (236.70): C, 55.82; H, 5.54; N, 23.67%. Found: C, 55.75; H, 5.39, N, 23.69%.
4-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indole (3b). Yield 0.67 g (67%); mp 156-
157 °C (dichloromethane/ethyl acetate, 1:1 v/v → ethyl acetate); IR (KBr, cm−1) νmax 3402,
3172, 2955, 2877, 1643, 1620, 1501, 1227, 969, 739; 1H NMR (200 MHz, CDCl3) δ 3.40 (br
s, 4H), 4.10-5.20 (br signal, 1H), 6.0-6.70 (br signal, 1H), 6.50 (d, J = 3.3 Hz, 1H), 6.69-6.78
(m, 1H), 7.04-7.09 (m, 3H); 13C NMR (50 MHz, CDCl3) δ 42.8, 94.8, 103.8 (d, J = 19.0 Hz),
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105.7 (d, J = 3.4 Hz), 115.3 (d, J = 22.9 Hz), 122.2 (d, J = 8.0 Hz), 127.4, 137.5 (d, J = 11.4
Hz), 156.5 (d, J = 247 Hz), 166.5. Anal. Calcd for C11H11FN4 (218.22): C, 60.55; H, 5.08; N,
25.67%. Found: C, 60.41; H, 5.19, N, 25.58%. Hydrochloride of 3b. Mp 181-182 °C; IR
(KBr, cm−1) νmax 3086, 2922, 1656, 1622, 1492, 1236, 974, 743. Anal. Calcd for
C11H12ClFN4 (254.68): C, 51.88; H, 4.75; N, 21.99%. Found: C, 52.03; H, 4.87, N, 22.18%.
5-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indole (3c). Yield 0.46 g (46%); mp 58-
60 °C (dichloromethane/ethyl acetate, 1:1 v/v → ethyl acetate); IR (KBr, cm−1) νmax 3397,
1
3171, 2954, 2878, 1644, 1615, 1427, 1283, 1211, 1117, 796, 715; H NMR (200 MHz,
CDCl3) δ 3.42 (br s, 4H), 4.0-5.70 (br signal, 2H), 6.37 (d, J = 3.14 Hz, 1H), 6.91 (dt, J1 = 2.5
Hz, J2 = 9.1 Hz, 1H), 7.10 (d, J = 3.2 Hz, 1H), 7.16-7.25 (m, 2H); 13C NMR (50 MHz,
CDCl3) δ 42.8, 98.7 (d, J = 4.9 Hz), 105.4 (d, J = 23.6 Hz), 109.9 (d, J = 20 Hz), 110.3 (d, J =
2.9 Hz), 126.3 (d, J = 10.4 Hz), 128.7, 131.2, 158.0 (d, J = 233.4 Hz), 166.4. Anal. Calcd for
C11H11FN4 (218.22): C, 60.55; H, 5.08; N, 25.67%. Found: C, 60.63; H, 5.25, N, 25.69%.
Hydrochloride of 3c. Mp 206-208 °C; IR (KBr, cm−1) νmax 3291, 3132, 3086, 2891, 1655,
1633, 1473, 1241, 1125, 946, 868, 809, 756 Anal. Calcd for C11H12ClFN4 (254.68): C, 51.88;
H, 4.75; N, 21.99%. Found: C, 51.73; H, 4.71, N, 22.08%.
5-Chloro-1-[(imidazolidin-2-yl)imino]-1H-indole (3d). Yield 0.54 g (50%); mp
108-110 °C (ethyl acetate/methanol, 9.5:0.5 v/v, crystallization from ethyl acetate); IR (KBr,
cm−1) νmax 3405, 3391, 3133, 2965, 28757, 1643, 1604, 1498, 1447, 1283, 1213, 1076, 1042,
1
904, 792, 754, 711; H NMR (500 MHz, DMSO-d6) δ 3.35 (br s, 4H), 6.16 (br s, 1H), 6.34
(d, J = 2.9 Hz, 1H), 6.68 (br s, 1H), 7.04 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.24
13
(d, J = 2.9 Hz, 1H), 7.55 (s, 1H); C NMR (50 MHz, DMSO-d6) δ 42.5, 97.7, 111.3, 119.5,
120.6, 123.1, 127.0, 129.7, 133.1, 166.0. Anal. Calcd for C11H11ClN4 (234.69): C, 56.30; H,
4.72; N, 23.87%. Found: C, 56.24; H, 4.65; N, 23.80%. Hydrochloride of 3d. Mp 204-207
°C (crystallization from ethanol/diethyl ether, 1:5 v/v); IR (KBr, cm−1) νmax 3120, 3014,
2916, 1644, 1623, 1456, 1294, 1226, 1080, 1047, 792, 753, 720. Anal. Calcd for C11H12Cl2N4
(271.15): C, 48.73; H, 4.46; N, 20.66%. Found: C, 48.64; H, 4.31; N, 20.35%.
1-[(Imidazolidin-2-yl)imino]-5-methyl-1H-indole (3e). Yield 0.37 g (38%); mp 63-
65 °C (ethyl acetate/methanol, 9.5:0.5 v/v); IR (KBr, cm−1) νmax 3387, 3173, 2871, 1644,
1
1499, 1473, 1328, 1283, 1211, 1077, 794, 756, 711; H NMR (500 MHz, DMSO-d6) δ 2.36
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(s, 3H), 3.26-3.29 (m, 2H), 3.37-3.40 (m, 2H), 5.92 (s, 1H), 6.22 (d, J = 2.6 Hz, 1H), 6.54 (s,
1H), 6.87 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 2.6 Hz, 1H), 7.27 (s, 1H);
13C NMR (125 MHz, DMSO-d6) δ 21.9, 42.6, 43.3, 97.6, 110.1, 120.4, 122.7, 126.7, 127.1,
128.3, 133.5, 167.0. Anal. Calcd for C12H14N4 (214.27): C, 67.27; H, 6.59; N, 26.15%.
Found: C, 67.18; H, 6.47; N, 25.97%. Hydrochloride of 3e. Mp 224-226 °C (crystallization
from acetonitrile); IR (KBr, cm−1) νmax 3132, 3079, 3025, 2926, 1651, 1630, 1471, 1327,
1299, 1224, 1085, 800, 762, 727. Anal. Calcd for C12H15ClN4 (250.73): C, 57.48; H, 6.03; N,
22.35%. Found: C, 57.39; H, 5.97; N, 22.34%.
6-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indole (3f). Yield 0.46 g (46%); mp 118-
120 °C (dichloromethane/ethyl acetate, 1:1 v/v → ethyl acetate); IR (KBr, cm−1) νmax 3395,
1
3169, 2955, 2877, 1643, 1614, 1483, 1329, 1284, 1218, 1166, 943, 800, 710; H NMR (200
MHz, CDCl3) δ 3.44 (br s, 4H), 4.10-6.20 (br signal, 2H), 6.40 (d, J = 3.3 Hz, 1H), 6.83 (dt,
J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 6.97 (dd, J1 = 2.1 Hz, J2 = 9.9 Hz, 1H), 7.05 (d, J = 3.1 Hz, 1H),
7.48 (dd, J1 = 5.2 Hz, J2 = 8.7 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 42.8, 95.7 (d, J = 26.5
Hz), 99.2, 108.1 (d, J = 24.8 Hz), 121.6 (d, J = 10.1 Hz), 122.7, 127.8 (d, J = 3.7 Hz), 134.6
(d, J = 12.4 Hz), 160.0 (d, J = 237.6 Hz), 166.3. Anal. Calcd for C11H11FN4 (218.22): C,
60.55; H, 5.08; N, 25.67%. Found: C, 60.71; H, 5.14, N, 25.74%. Hydrochloride of 3f. Mp
214-216 °C; IR (KBr, cm−1) νmax 3128, 2921, 1661, 1621, 1481, 1210, 943, 842, 801. Anal.
Calcd for C11H12ClFN4 (254.68): C, 51.88; H, 4.75; N, 21.99%. Found: C, 51.99; H, 4.92, N,
21.79%.
7-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indole (3g). Yield 0.39 g (39 %); 135-
137 °C (dichloromethane/ethyl acetate, 1:1 v/v → ethyl acetate); IR (KBr, cm−1) νmax 3403,
3172, 2955, 2876, 1643, 1619, 1571, 1500, 1283, 1237, 1084, 782, 715; 1H NMR (200 MHz,
CDCl3) δ 3.47 (br s, 4H), 4.10-5.0 (br signal, 1H), 5.50-6.50 (br signal, 1H), 6.41 (t, J = 3.0
Hz, 1H), 6.75-6.97 (m, 2H), 7.04 (d, J = 3.0 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H); 13C NMR (50
MHz, CDCl3) δ 42.9, 99.5, 107.1 (d, J = 17.3 Hz), 116.7 (d, J = 3.6 Hz), 119.2 (d, J = 6.4
Hz), 122.6 (d, J = 9.2 Hz), 128.9, 130.6 (d, J = 5.0 Hz), 149.9 (d, J = 245.8 Hz), 166.9. Anal.
Calcd for C11H11FN4 (218.22): C, 60.55; H, 5.08; N, 25.67%. Found: C, 60.61; H, 5.28, N,
25.51%. Hydrochloride of 3g. Mp 201-203 °C; IR (KBr, cm−1) νmax 3235, 3076, 2724, 1657,
1616, 1581, 1485, 1288, 1244, 784, 717, 670. Anal. Calcd for C11H12ClFN4 (254.68): C,
51.88; H, 4.75; N, 21.99%. Found: C, 52.14; H, 4.41, N, 21.92%.
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7-Chloro-1-[(imidazolidin-2-yl)imino]-1H-indole (3h). Yield 0.49 g (45%);
colourless oil (dichlorometane → dichloromethane/ethyl acetate, 1:1 v/v → ethyl acetate); IR
1
(liquid film, cm–1) νmax 3404, 3167, 2873, 1642, 1498, 1278, 1192, 935, 782, 717; H NMR
(200 MHz, CDCl3) δ 3.33-3.62 (m, 4H), 4.32 (br s, 1H), 6.42-6.43 (m, 1H), 6.50 (br s, 1H),
6.94 (t, J = 7.7 Hz, 1H), 7.07-7.09 (m, 2H), 7.47 (dd, J1 = 7.7 Hz, J2 = 0.8 Hz, 1H); 13C NMR
(50 MHz, CDCl3) δ 42.8, 98.9, 116.4, 119.5, 122.8, 129.2, 129.4, 167.2. Anal. Calcd for
C11H11ClN4 (234.69): C, 56.30; H, 4.72; N, 23.87%. Found: C, 56.41; H, 4.68; N, 23.80%.
Hydrochloride of 3h. Mp 231-233 °C; IR (KBr, cm–1) νmax 3094, 3005, 2903, 2804, 1655,
1478, 1272, 1194, 1147, 793. Anal. Calcd for C11H12Cl2N4 (271.15): C, 48.73; H, 4.46; N,
20.66%. Found: C, 48.85; H, 4.32; N, 20.53%.
1-[(Imidazolidin-2-yl)imino]-7-methoxy-1H-indole (3i). Yield 0.65 g (61%); mp
179-180 °C (ethyl acetate/methanol, 9.5:0.5 v/v); IR (KBr, cm−1) νmax 3392, 3169, 2873,
1
1645, 1608, 1572, 1501, 1479, 1284, 1257, 1194, 1093, 1082, 1031, 965, 783, 711, 690; H
NMR (200 MHz, CDCl3) δ 3.45 (s, 4H), 3.88 (s, 3H), 4.75 (br s, 2H), 6.37 (d, J = 3.0 Hz,
1H), 6.59 (d, J = 7.5 Hz, 1H), 6.95 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 3.0 Hz, 1H), 7.24 (d, J =
7.5 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 43.4, 56.7, 99.2, 103.4, 114.4, 119.9, 124.2,
128.4, 129.4, 147.6, 167.2. Anal. Calcd for C12H14N4O (230.27): C, 62.58; H, 6.13; N,
24.33%. Found: C, 62.47; H, 5.98, N, 24.29%. Hydrobromide of 3i. Mp 150-152 °C; IR
(KBr, cm−1) νmax 3135, 2906, 1656, 1631, 1582, 1486, 1463, 1443, 1385, 1338, 1286, 1257,
1097, 1084, 1050, 1007, 966, 784, 721, 690. Anal. Calcd for C12H15BrN4O: C, 46.32; H,
4.86; N, 18.00%. Found: C, 46.02; H, 5.12; N, 17.73%.
Synthesis of 1-[(imidazolidin-2-yl)imino]-7-methyl-1H-indole (3j). Compound 3j
was obtained according to the method described by F. Sączewski et al. (16). Step 1. To a
stirred solution of 1-amino-7-methyl-1H-indole (2j) (0.40 g, 2.7 mmol), N,N′-bis(tert-
butoxycarbonyl)imidazolidine-2-thione (1.23 g, 4.06 mmol), and triethylamine (0.97 g, 1.33
mL, 9.6 mmol) in DMF (4 mL) was added HgCl2 (1.1 g, 4.06 mmol) at 0 °C. The reaction
mixture was stirred for an additional 20 min at 0 °C, and then for 5 days at room temperature.
The resulting dark-grey reaction mixture was diluted with ethyl acetate (40 mL) and filtered
through a celite pad. The filtrates were washed with brine (3x) and water (1x), dried over
MgSO4, and finally concentrated under vacuum. The viscous residue was subjected to
preparative thin layer chromatography eluting with hexane/dichloromethane (1:5 v/v)
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followed by dichloromethane to afford 0.30 g 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-
yl)]imino}-7-methyl-1H-indole (4). Yield 26%; mp 128-130 °C; IR (KBr, cm−1) νmax 2980,
2957, 2923, 1740, 1716, 1652, 1475, 1455, 1390, 1309, 1252, 1153, 1139, 988, 845, 787,
765, 717; 1H NMR (200 MHz, DMSO-d6) δ 0.83 (s, 9H), 1.50 (s, 9H), 2.70 (s, 3H), 3.79-3.87
(m, 4H), 6.38 (d, J = 3.3 Hz, 1H), 6.83-6.89 (m, 2H), 7.29-7.32 (m, 1H), 7.41 (d, J = 3.3 Hz,
1H). Anal. Calcd for C22H30N4O4 (414.50): C, 63.75; H, 7.30; N, 13.52%. Found: C, 63.89;
H, 7.46; N, 13.49%. The obtained compound 4 was used for the next step without further
purification. Step 2. A solution of the Boc-protected imidazolidine 4 (0.22 g, 0.72 mmol) in
trifluoroacetic acid in dichloromethane (4 mL, 1:1, v/v) was stirred for 2 h at room
temperature, and then the solvent and excess of trifluoroacetic acid were evaporated under
reduced pressure. The oily residue was treated with water, made alkaline with 5% aqueous
NaOH solution (pH 10-10.5), and the mixture was extracted with dichloromethane (3 x 15
mL). The combined organic layers were dried over MgSO4, filtered and evaporated to
dryness. The crude product 3j thus obtained was purified by preparative thin layer
chromatography eluting with hexane/dichloromethane (9.5:0.5 v/v). The free base 3j was
then converted into hydrobromide salt by adding 48% hydrobromic acid solution to the
solution of 1-[(imidazolidin-2-yl)imino]-7-methyl-1H-indole (3j) in methanol.
1-[(Imidazolidin-2-yl)imino]-7-methyl-1H-indole (3j). Yield 0.07 g (44%); mp 61-
64 °C; IR (KBr, cm−1) νmax 3392, 3185, 2953, 2877, 1644, 1499, 1457, 1283, 1076, 1042,
781, 718; 1H NMR (500 MHz, CDCl3) δ 2.64 (s, 3H), 3.37 (br s, 2H), 3.50 (br s, 2H), 4.58 (br
signal, 2H), 6.42-6.43 (m, 1H), 6.90 (d, J = 6.8 Hz, 1H), 6.95-6.98 (m, 1H), 7.00 (d, J = 2.9
Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H); 13C NMR (50 MHz, CDCl3) 18.7, 43.2, 99.3, 119.1, 119.7,
122.1, 124.1, 127.3, 128.2, 133.4, 167.1. Anal. Calcd for C12H14N4 (214.27): C, 67.27; H,
6.59; N, 26.15. Found: C, 67.38; H, 6.31; N, 25.87%. Hydrobromide of 3j. Mp: 189-192 °C
(crystallization from ethanol/diethyl ether, 1:5 v/v); IR (KBr, cm−1) νmax 3190, 3095, 2925,
1654, 1606, 1458, 1384, 1278, 1214, 1078, 782, 721. Anal. Calcd for C12H15BrN4 (295.18):
C, 48.83; H, 5.12; N, 18.98%. Found: C, 48.67; H, 4.93; N, 18.85%.
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Pharmacological methods
Radioligand binding assays
I1-imidazoline site binding assay. Kidneys were obtained post mortem from male
Sprague-Dawley rats (250-280 g) and crude P2 membranes prepared according to the
methods of Lione et al. (28). Binding of [3H]clonidine (3 nM, Perkin-Elmer) was investigated
in the presence of 10 μM rauwolscine to preclude radioligand binding to α2-ARs. The
specific component was defined by 10 μM rilmenidine; under these conditions, the site
labeled represents a model of the central I1 binding site (29). Membrane aliquots (400 μl, 0.2-
0.5 mg protein) were incubated with 11 concentrations of the test compounds over the range
0.1 nM-100 μM. Incubations were carried out in 50 mM Tris-HCl buffer (pH 7.4) at room
temperature for 45 min. Bound radioligand and free radioactivity were separated by rapid
filtration through pre-soaked (0.5% polyethyleneimine) glass-fibre filters (Whatman GFB).
Trapped radioligand was determined by liquid scintillation counting and the data were
analyzed with GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego,
CA, USA) to yield IC50 values (the concentration of tested ligand that displaces 50% of
specifically bound [3H]clonidine).
α1-ARs, α2-ARs and I2-binding site assays. Brains were obtained post mortem from
male Sprague-Dawley rats (250-280 g) and crude P2 membranes were prepared (28).
Membrane aliquots (400 μl, 0.2-0.3 mg protein) were incubated with 11 concentrations of the
tested compounds over the range 0.1 nM -100 μM in the presence of the selective I2 binding
site radioligand [3H]-2BFI (2-(2-benzofuranyl)-2-imidazoline) (30) (1 nM), the α1-AR
antagonist radioligand [3H]prazosin (1 nM) or the α2-AR antagonist radioligand
[3H]RX821002 (2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole)
(1 nM) in a final volume of 500 μl. Non-specific binding was determined using 10 μM
BU224 (2-(4,5-dihydroimidazol-2-yl)quinoline) (31) for I2 binding, 10 μM phenylephrine for
α1-ARs and 10 μM rauwolscine to define α2-AR binding. Incubations were performed in
triplicate at room temperature and were allowed to reach equilibrium (45 min). Bound and
free radioactivity were separated by rapid filtration through pre-soaked (0.5%
polyethyleneimine) glass-fibre filters (Whatman GF/B). Filters were then washed twice with
5 ml of ice-cold buffer and membrane-bound radioactivity remaining on the filters was
determined by liquid scintillation counting. The data were analyzed by iterative non-linear
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regression curve fitting procedures with GraphPad Prism version 4.03 for Windows
(GraphPad Software, San Diego, CA, USA). Each experiment was analyzed individually and
equilibrium dissociation constants (Ki) were determined by the method of Cheng and Prusoff
(32). The resulting values are given as the means SEM (standard error of measurement) of
three or four separate experiments.
In vivo studies mean arterial blood pressure (MAP) and heart rate (HR) in rats
Male Wistar rats, weighing 200-290 g were purchased from the Animal House of the
Medical University of Gdańsk, Poland. All in vivo experiments were approved by the Local
Ethical Committee on Animal Experiments. The animals were fed commercial rodent chow
(Labofeed-B, Poland). Tap water was available ad libitum. Rats were anesthetized by i.p.
injection of thiopental (Sandoz, Austria) at 70 mg/kg body weight and anesthesia was
maintained by thiopental supplementation (30 μg/kg/min) during the experiment. The
animals were placed on a heated table, and body temperature was maintained between 36 and
37 °C. Tracheotomy was performed. Catheters were inserted into the carotid artery for
monitoring of MAP and HR, into a jugular vein for infusions, and into the bladder for free
diuresis. After all surgical procedures, a 40 min recovery period was allowed to establish a
stable baseline. The rats were infused with isotonic saline (Fresenius Kabi, Poland)
supplemented with thiopental at a rate of 1.2 ml/h. After 40 min of saline infusion, the tested
compound was administered as a 100 μl bolus through the venous catheter at doses of either
0.01 or 0.1 mg/kg depending on cardiovascular potency of a tested compound. The time of
administration of a compound was assumed as “time 0”. MAP and HR were monitored
directly and sampled continuously at 100 Hz as described previously (33), using Biopac
Systems, Inc., Model MP 100 (Goleta, CA, USA). The recorded results were elaborated with
the help of the ACQKnowledge (Goleta, CA, USA) analysis system and were selected, scaled
and filtered to remove accidental signal disturbances. The recorded time domain transient
data are presented as graphs with the help of Excel (Microsoft, USA).
ANOVA was performed for ΔMAP and ΔHR, calculated as the difference in MAP
and in HR from baseline measurements (“time 0”) for each group, as described previously
(33). This allowed for direct comparison of responses to treatment between groups. Data
were analyzed by ANOVA for repeated measurements, using Statistica StatSoft software
(StatSoft, Inc., Tulsa, USA), after test compound or vehicle administration. When a treatment
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effect was significant, post hoc comparisons were performed using Fisher’s test. A value of
p<0.05 was considered statistically significant.
Metabolic stability
Stock solutions of studied compounds were prepared at concentration of 1 mM in
DMSO. Incubations were performed in the presence of 1 mM of NADPH (Sigma-Aldrich, St.
Louis, MO, USA) and 1 mg/mL of human liver microsomes (Sigma-Aldrich, St. Louis, MO,
USA) in potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried out in incubator
at 37 °C with agitation and started by addition of compound of interest at final concentration
of 1 µM (34,35). Directly after start of incubation and after 5, 15, 30, 45 and 60 min reaction
was ended by adding the equal volume of cold acetonitrile containing 1 μM of IS
(alprazolam). All samples were immediately centrifuged (10 min 10 000 rpm) and resulted
supernatant was directly analyzed or kept in −80 °C until LC-MS analysis. ln(A/AIS) was
plotted versus incubation time (A – peak area of compound of interest, AIS – peak area of
internal standard). In vitro metabolic half-life (t1/2) was calculated from the slope of the linear
regression, according to equation reported in (34,35).
LC-MS analysis was performed on an Agilent 1260 system coupled to SingleQuad
6120 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Ascentis Express
ES-CN column (2.1 x 100 mm, 2.7 μm, Sigma-Aldrich, St. Louis, MO, USA) was used in
reversed-phase mode with gradient elution starting with 5% of phase A (10 mM ammonium
formate buffer in water, pH 3) and 95% of phase B (10 mM ammonium formate in
acetonitrile-water mixture, 50:5 v/v, pH 3). The amount of phase B was linearly increased to
100% in 5 minutes. Total analysis time was 10 min at 25 °C, flow rate was 0.4 mL/min and
the injection volume was 10 μL. The mass spectrometer was equipped with electrospray
ionization source and ionization mode was positive. Mass analyzer was set individually to
each compound to detect pseudomolecular ions [M+H+]. MSD parameters of the ESI source
were as follows: nebulizer pressure 50 psig (N2), drying gas 13 mL/min (N2), drying gas
temperature 300 °C, capillary voltage 3.5 kV, fragmentor voltage 100 V.
Metabolic stability was additionally assessed by in silico technique proposed by
Zaretzki et al. based on a model build on molecular descriptors and trained using neural
networks (36).
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X-ray structure analysis
Diffraction data for single crystal of compound 3d were collected with an Oxford
Diffraction, XcaliburE diffractometer at 293(2) K. Using Olex-2 (37), the structure was
solved with SIR2004 (38) using direct methods and refined with ShelXL-2014 (39). H atoms
bonded to C were placed geometrically and refined as riding on their carriers. H atoms
bonded to N were freely refined with isotropic temperature factors.
Crystal data for C11H11ClN4 (3d) (M = 234.69 g/mol): monoclinic, space group P21/c (no.
14), a = 11.9693(6) Å, b = 9.1986(4) Å, c = 21.5853(11) Å, β = 105.513(5)°, V = 2290.0(2)
Å3, Z = 8, T = 293(2) K, μ(MoKα) = 0.311 mm-1, Dcalc = 1.361 g/cm3, 14010 reflections
measured (8.342° ≤ 2Θ ≤ 50.05°), 4016 unique (Rint = 0.0375, Rsigma = 0.0479) which were
used in all calculations. The final R1 was 0.0473 (I > 2σ(I)) and wR2 was 0.1038 (all data).
CCDC deposit number: CCDC 1479707.
Results and Discussion
Chemistry
The known 1-amino-1H-indoles 2a (19), 2b (22), 2c (23), 2d (20), 2e (21) and 2j (24)
as well as the novel derivatives 2f-i were prepared using previously described procedures
(20,23) (Scheme S1, Supporting Information). As shown in Scheme 1, 1-amino-1H-indoles
2a-i were then reacted with N-Boc-2-methylthio-4,5-dihydro-1H-imidazole (25) in acetic acid
to obtain, after deprotection of the formed intermediates A, the target free bases 3a-i (27).
The synthesis of the analogue 3j was accomplished by reacting 1-amino-1H-indole 2j with
N,N′-bis-Boc-imidazolidine-2-thione (26) in the presence of HgCl2, followed by cleavage of
the protecting group upon treatment of the Boc-protected compound 4 with TFA/CH2Cl2
(Scheme 1) (16).
For biological studies, compounds 3a-j were converted into water-soluble
hydrochlorides or hydrobromides with the use of methanolic or ethereal solution of
hydrochloride or hydrobromic acid solution, respectively.
Structures of the newly prepared compounds 3a-j were confirmed by C, H, N
elemental analyses, IR and NMR spectroscopic data as well as X-ray structure analysis of the
compound 3d (Figure 2).
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Single X-ray analysis of compound 3d confirmed that as expected, the imidazolidin-2-
imine tautomer exists in the crystalline phase. Moreover, comparison of the crystal structures
of indole 3d and 4-Cl-marsanidine of type D (16) revealed that both compounds in solid
phase adopt similar conformation, where benzazole and imidazolidin-2-imine fragments are
nearly perpendicular (dihedral angle of 87.04° and 80.75°, respectively).
As revealed by the data in Table 1, the tested compounds 3a-j were in general nearly
inactive at the I1 binding sites (Ki values in the range of 607–159000 nM), with the exception
of the 4-F-indole derivative 3b having moderate affinity for I1-receptors (Ki = 241 nM).
Similar results were obtained for imidazoline I2 receptors (Ki values ranging from 354 to
2708 nM).
On the other hand, the indole derivatives 3a-j were bound with moderate or high
affinity to α-ARs. Accordingly, the unsubstituted compound 3a showed good affinity for α1-
ARs (Ki = 70.2 nM) and high affinity for α2-ARs (Ki = 5.33 nM). However, placement of
substituents at positions C-4 or C-5 of the indole ring resulted in 3- to 12-fold decreases of
the binding properties (3b: α1Ki = 348 nM, α2Ki = 63.6 nM; 3c: α1Ki = 165 nM, α2Ki = 23.4
nM; 3d: α1Ki = 214 nM, α2Ki = 57.9 nM; 3e: α1Ki = 118 nM, α2Ki = 16.5 nM), while the 6-
F- substituted derivative 3f stayed equipotent to its unsubstituted counterpart 3a (α1Ki =
70.77 nM and α2Ki = 7.073 nM). Interestingly, within a series of 7-substituted derivatives, the
7-F, 7-Cl and 7-CH3 analogues 3g, 3h and 3j retained comparably high affinity for α2-ARs,
and displayed the following Ki values: 12 nM for 3g (7-F), 9.95 nM for 3h (7-Cl) and 12.09
nM for 3j (7-CH3), whereas their potencies for α1-ARs were much more different. It
appeared that the 7-Cl congener 3h was 4-times more active at α1-ARs than 7-F- and 7-CH3-
substituted indoles 3g and 3j (Ki of 38.85, 167 and 166 nM, respectively). On the other hand,
placement of OCH3 group at position 7 (3i) reduced both the α1- and α2-AR interactions (Ki =
807 and 151 nM, respectively). The data presented in Table 2 indicate that the presence of a
small substituent at position 7 characterized by positive lipophilic contribution is suitable for
high affinity at α2-ARs (3g: 7-F π = 0.14; 3h: 7-Cl π = 0.71; 3j: 7-CH3 π = 0.56) (40).
Moreover, 7-Cl substituent, which exhibits high lipophilicity as well as relatively high
electronegativity (41), is responsible for binding affinity to α1-ARs.
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In comparison to the previously investigated indazole analogues D (Table 1, values
given in parentheses) (16,17,27), the indoles 3a-j displayed 2- to 7-fold increases in receptor
binding affinity for α2-ARs with different degrees of selectivity for α2-ARs versus I1 binding
sites (I1/α2 selectivity ratios ranging from 4 to 9636). The highest difference in potencies at
α2-ARs and I1-imidazoline receptors was showed by unsubstituted indole 3a as well as 5-
CH3- and 7-F-substituted derivatives 3e and 3g (I1/α2 selectivity ratio of 1300, 9636 and
1269, respectively). It is worth noting that the investigated indole derivatives 3 displayed
relatively low selectivity for α2-ARs versus α1-ARs (α1/α2 selectivity ratios in the range of
3.7–14, Table 1).
In vivo cardiovascular effects
To determine the effects of the displacement of the indazole ring in 1-[(imidazolidin-
2-yl)imino]-1H-indazoles D with the indole ring on the cardiovascular properties, the newly
prepared compounds 3 were evaluated in anesthetized rats by directly measuring changes in
blood pressure and heart rate after intravenous administration at doses of either 0.01 or 0.1
mg/kg depending on cardiovascular potency of a tested compound and using previously
described procedure (33). The post-infusion data were compared to baseline values and are
presented as ΔMAP and ΔHR in Table 3.
First, most of the tested compounds 3 elicited a biphasic effect on blood pressure after
intravenous administration that is transient hypertension followed by a prolonged
hypotension effect. The observed changes were similar to those obtained in the case of
marsanidine-like ligands D (16,17) and might be due to the stimulation of peripheral α1-ARs
or vascular α2-ARs (a short-term increase in blood pressure) followed by activation of central
α2-ARs (a long-lasting hypotensive effect) (3,42,43). The pressure phase was accompanied
by a marked bradycardia, which persisted through the longer hypotensive phase. The above
cardiovascular effects elicited by 3a, 3f and 3g are also presented in Figures S1 and S2
(Supporting Information).
In our previous SAR analysis on marsanidine-like ligands D (16,17,44) it has been
revealed that substitution at the C-7 atom of the indazole ring with chlorine, fluorine or
methyl group led to compounds with high cardiovascular properties, wherein the greatest
hypotensive activity was noted for the congener bearing a fluorine that is 7-F-marsanidine of
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type D (17). A similar trend was found for indoles 3 as 7-F-substituted derivative 3g
displayed the largest hemodynamic effects in the respective 3a-j series (Table 3). It should be
mentioned that hypotensive and negative chronotropic effects of 3g administered at a dose of
0.01 mg/kg (ΔMAP = −65.0 mmHg, ΔHR = −148 bpm) were larger than those of 7-F-
marsanidine at a higher dose of 0.1 mg/kg (ΔMAP = −59.4 mmHg, ΔHR = −128 bpm) (17).
In comparison to the indazole ring system the indole moiety is more lipophilic (logPindole 2.14
versus logPindazole 1.77) (40). Therefore it appears that the more lipophilic nature and the
higher α2-binding affinity of 7-F-indole 3g with respect to that of 7-F-indazole D may
facilitate its ability to penetrate the brain-blood-barrier and account for its more efficacious
activity than 7-F-indazole D, which is a partial α2-AR agonist (17,18). In addition, under
identical experimental conditions, changes of ΔMAP induced by 3g were much greater as
compared to those of 3a and 3f (Table 3, Figure S1) even though they showed higher activity
at α2-ARs (Table 1). Similarly, 3g administration resulted in a greater decrease of ΔHR in
comparison to that of 3a and 3f (Table 3, Figure S2).
As expected, 7-Cl and 7-CH3-substitued derivatives 3h and 3j with high affinity to α2-
ARs also displayed efficacious hypotensive effects at a dose of 0.1 mg/kg (ΔMAP = –42.3
and –40.2 mmHg, respectively), however slightly lower than those of their 7-Cl (ΔMAP = –
49.6 mmHg) (17) and 7-CH3 (ΔMAP = –43.5 mmHg) (16) indazole analogues D at the same
dose. Moreover, intravenous administration of these compounds elicited a significant
bradycardic effects (ΔHR = −124.6 and −158.2 bpm, respectively). On the other hand, 5-
CH3-substituted indole 3e with similar receptor affinity profile as compared to 7-CH3
congener 3j and the highest α2- versus I1-selectivity of 9636 (Table 1) exhibited much less
pronounced cardiovascular activity (ΔMAP = –21.8 mmHg, ΔHR = −94.8 bpm). It should be
pointed out that 4-F congener 3b, characterized by good affinity for α2-ARs and moderate
affinity for I1 imidazoline binding sites, showed greater hypotensive activity (ΔMAP = –51.7
mmHg) than that of 3h and 3j. Therefore, both I1-imidazoline and α2-adrenergic receptors
might be possible mediators of the observed hypotensive effect of the tested compound 3b
(10-11,16). Finally, much lower cardiovascular activity was observed for 5-Cl and 7-OMe
derivatives 3d and 3i (3d: ΔMAP = –11.4 mmHg, ΔHR = −39.9 bpm; 3i: ΔMAP = –16.6
mmHg, ΔHR = −66.0 bpm), although they displayed relatively moderate affinity for α2-ARs
(Ki = 57.9 and 151 nM, respectively).
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It is generally assumed that a short-time hypertensive phase followed by a long lasting
decrease in blood pressure with accompanying bradycardia is a common effect of the
centrally acting α2-AR agonists, such as clonidine (45,46). The bradycardia induced by α2-
AR agonists relates to their central sympatholytic action with unopposed vagal tone,
presynaptic inhibition of noradrenaline release, or direct vagotonic effect (6,47). Recently,
our studies concerning cardiovascular effects of marsanidine and 7-CH3-marsanidine in
vagotomised rats have revealed that cardiovascular activities of these compounds are not due
to vagal nerves, nevertheless, vagotomy enhanced sensitivity of the sympathetic pathways for
the tested compounds (48). Hence, we propose that the long-lasting heart rate decrease and
hypotensive effect of the tested imidazoline-containing indoles 3 result from their central
sympathetic depression. However, taking into account their relatively high or moderate
affinities to α1-ARs, a contribution of α1-ARs to cardiac function can not be excluded (49-
51).
Metabolic stability
Metabolic stability assessed in the presence of human liver microsomes and NADPH
as a cofactor showed that the studied compounds 3a, 3f and 3h were stable under these
conditions. After 60 min of incubation ca 90% of studied compounds remained unchanged.
Additionally, XenoSite, in silico model proposed by Zaretzki et al. (36) showed that the
studied derivatives 3a, 3f and 3h are characterized by low vulnerability to undergo metabolic
reactions mediated by cytochrome P-450 as depicted in Figure S3 (Supporting Information).
Our results indicate high stability of the tested compounds, especially for first phase
metabolism.
Conclusion
The displacement of the indazole ring in marsanidine-like ligands D (16,17) with the
indole moiety results in compounds 3 with higher affinity at α2-ARs and high or moderate
affinity at α1-ARs. The pronounced hypotensive and bradycardic effects of novel α-
adrenergic ligands 3g, 3h and 3j are in agreement with our previous observation that C-7
substitution of the azaaromatic ring, such as indazole (16,17,44), plays a crucial role in
cardiovascular activities of this class of imidazoline-containing compounds. Similar
hemodynamic activity profile of marsanidine-like compounds D and indole derivatives 3
suggests that the hypotensive effect of indoles 3 is mediated by central α2-ARs. In
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contradistinction to our earlier observation that the bioisosteric 1-[(imidazolin-2-
yl)methyl]indazoles (B) and 1-[(imidazolin-2-yl)methyl]indoles (C) exert different
hemodynamic effects (15), the present studies indicate that 1-[(imidazolidin-2-
yl)imino]indazoles (D) and their indole analogues 3 produce similar cardiovascular effects.
Moreover, the results of in vitro metabolic stability studies as well as in silico model revealed
that indoles 3 are not vulnerable to rapid first phase oxidative metabolism.
Conflict of interest
The authors declare no conflict of interest.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Appendix S1. Preparation and analytical data of 1-amino-1H-indoles 2f-i.
Scheme S1. Synthesis of 1-amino-1H-indoles 2a-j.
Figure S1. Effect of 0.01 mg/kg of 3a, 3f and 3g on ΔMAP (calculated as the difference of
MAP between sequential measurement and time 0 of the experiment) in rats. The lines
represent the mean values of ΔMAP for four or five experiments. Comparison were made
using ANOVA with repeated measures and Fisher’s test. Significance (*) p < 0.001 was
found for comparisons of the following: 3f and 3g versus control group; 3a and 3f versus 3g.
Significance (#) p < 0.01 was found for comparison of 3a versus control group. Significance
(&) p < 0.05 was found for comparison of 3a and 3f.
Figure S2. Effect of 0.01 mg/kg of 3a, 3f and 3g on ΔHR (calculated as the difference of HR
between sequential measurement and time 0 of the experiment) in rats. The lines represent
the mean values of ΔHR for four or five experiments. Comparison were made using ANOVA
with repeated measures and Fisher’s test. Significance (*) p < 0.001 was found for
comparisons of the following: 3a and 3f versus 3g and 3g versus control group. Significance
(#) p < 0.01 was found for comparison of 3a and 3f versus control group.
Figure S3. Output from XenoSite in silico tool for prediction of sites of metabolism. Color
scale bar shows probability to undergo metabolic reactions mediated by cytochrome P-450
isoforms present in human liver microsomes.
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Figures Legends
1. Figure 1. Known imidazoline α-adrenoceptor ligands A-D and the newly designed 1-
[(imidazolidin-2-yl)imino]-1H-indoles 3.
2. Figure 2. Molecule structure of 3d with displacement ellipsoids drawn at the 50%
probability level.
Scheme Legend
1.
2. Scheme 1. Synthesis of 1-[(imidazolidin-2-yl)imino]-1H-indole derivatives 3a-j.
Table Legends
1. Table 1. Binding affinity data for the prepared indoles 3a-j and selected indazoles of
type D (values given in parentheses)
2. Table 2. Hydrophobic constants (π) and Pauling electronegativities (χ) for substituents
(R) of indole derivatives and binding affinities of 3g-j to α-adrenergic receptors
3. Table 3. Effects of the prepared indoles 3a-j at 0.01 or 0.1 mg/kg i.v. on mean arterial
blood pressure (MAP) and heart rate (HR) in anesthetized rats
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Table 1. Binding affinity data for the prepared indoles 3a-j and selected indazoles of type D
(values given in parentheses)
Compound
Binding affinitiesa
Selectivity
ratio
α1 Ki (nM)b
α2 Ki (nM)b
α1/α2 I1/α2
No
R
I1 IC50 (nM)c
I2 Ki (nM)b
3a (D:
H
70.2 (n = 1)e
5.33 (n = 1)e
(14.05)
6930 (n = 1)e
(54500)
1840 (n = 1)e
(16900)
13
5.5
7
1300
(3879)
13a)d
(nd)g
3b (D: 4-F
4 (35)
348 146.5
(3807)
63.6 8.561
(416)
241 7.024
(14776)
2127 351.8
(6177)
6a)g
3c (D: 5-F
964
(1.4)
165 25.48
(2320)
23.4 1.986
(64.33)
22557 15338 335.7 103.5
(91.65) (5703)
6b)g
3d (D: 5-Cl
214 (n = 1)e
57.9 (n = 1)e
(114.2)
16.5 (n = 1)e
(44.2)
17300 (n = 1)e 1150 (n = 1)e
3.7
7
299
(149)
13h)d
(nd)f
(16980)
(20620)
3e (D: 5-
118 (n = 1)e
159000 (n =
354 (n = 1)e
(7159)
9636
(459)
13g)d
CH3
(nd)f
1)e (20310)
3f (D: 6-F
10
14
4
745
(789)
70.77 9.51
(918.3)
7.073 0.3886 5270 2659
422.7 43.21
(21967)
6c)g
(26.20)
(20675)
3g (D: 7-F
1269
(250)
167 26.1
(1625)
12 0.7371
15230 6203
1087 432.9
(348833)
6d)g
(30.97)
(7740)
3h (D)h 7-Cl
95
38.85 5.88
9.95 2.52
949.7 532.8
2708 1022
(1544)
(1010)
(30.3)
(46800)
(15000)
7-
OCH3
807 (n = 1)e
151 (n = 1)e
607 (n = 1)e
748 (n = 1)e
5.3
13.7
4
3i
3j (D: 7-
591.6
(7.2)
166.0 47.7
12.09 2.37
(53.5)
7152 6775
(387)
2536 950.5
(2520)
13k)d
CH3
(nd)f
a Values given are means SEM of three or four independent experiments.
b
Ki affinity values for α1-adrenoceptors, α2-adrenoceptors, and I2 imidazoline binding sites were assessed by
measuring the ability of the tested compounds to compete with [3H]prazosin, [3H]RX821002 or [3H]2BFI
binding to rat brain membranes.
c
Molar concentration of the tested compounds that displaces 50% of specifically bound [3H]clonidine in rat
kidney membranes in the presence of rauwolscine (I1 imidazoline binding sites).
d The results have been published earlier in Sączewski et al. (16).
e
n: number of experiments.
f nd: not determined.
g The results have been published earlier in Wasilewska et al. (17).
h The results have been published earlier in Sączewski et al. (27).
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Table 2. Hydrophobic constants (π) and Pauling electronegativities (χ) for substituents (R) of
indole derivatives and binding affinities of 3g-j to α-adrenergic receptors
Compound
πa
χb
α1Ki (nM) α2Ki (nM)
No
R
F
0.14
0.71
−0.02
0.56
4.0
3.0
2.7
2.3
167
12
3g
3h
3i
9.95
151
12.09
Cl
38.85
807
OCH3
CH3
166
3j
a According to ref. 40.
b According to ref. 41.
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Table 3. Effects of the prepared indoles 3a-j at 0.01 or 0.1 mg/kg i.v. on mean arterial blood pressure (MAP) and heart rate (HR) in anesthetized
rats
ne
a,d
b,d
c,d
Compound
Dose
(mg/kg)
p
p
p
+ΔMAPmax
(mmHg)
–ΔMAPmax
(mmHg)
–ΔHRmax
(bpm)
No
R
H
0.01
0.1
<0.03
<0.001
<0.003
–
<0.003
<0.001
<0.001
–
<0.001
<0.001
<0.001
<0.005
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
–
5
4
4
4
7
4
4
5
5
5
5
3a
21.1 7.0
35.8 5.5
25.3 5.4
8.5 4.1
26.6 4.7
14.6 3.4
15.3 1.6
26.6 5.2
0.5 0.3
18.4 0.8
3.4 0.9
–24.0 3.8
–51.7 5.9
–28.4 4.3
–11.4 1.9
–21.8 0.9
–37.2 2.1
–65.0 1.1
–42.3 2.9
–16.6 1.3
–40.2 3.9
–6.2 1.7
–80.4 12.6
–136.3 17.4
–110.8 12.6
–39.9 1.5
–94.8 5.6
–87.4 7.7
–148 4.2
4-F
3b
3c
5-F
0.1
5-Cl
5-CH3
6-F
0.1
3d
3e
0.1
<0.002
<0.009
<0.001
<0.002
–
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
–
0.01
0.01
0.1
3f
7-F
3g
7-Cl
7-OCH3
7-CH3
3h
3i
–124.6 14.0
–66.0 7.5
–158.2 13.0
–22.9 3.6
0.1
0.1
<0.001
–
3j
controlf
a The maximal hypertensive effect [mmHg] of a compound observed during 60 min after injection.
b The maximal hypotensive effect [mmHg] of a compound observed during 60 min after injection.
c The maximal negative chronotropic effect [bpm] of a compound observed during 60 min after injection.
d Values given are means SEM from n independent experiments.
e Number of experiments.
f Saline vehile injection. Comparisons were performed using the Student’s t test; p significance versus control.
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