Application of "hydrogen bonding interaction" in new drug development: Design, synthesis, antiviral activity, and sars of thiourea derivatives

Article abstract of DOI:10.1021/jf505355r

A series of simple thiourea derivatives were designed based on the structure of natural product harmine and lead compound and synthesized from amines in one step. The antiviral activity of these thiourea derivatives was evaluated. Most of them exhibited significantly higher anti-TMV activity than commercial plant virucides ribavirin, harmine, and lead compound. The hydrogen bond was found to be important but not the more the better. The optimal compound (R,R)-20 showed the best anti-TMV activity in vitro and in vivo (in vitro activity, 75%/500 a??g/mL and 39%/100 a??g/mL; inactivation activity, 71%/500 a??g/mL and 35%/100 a??g/mL; curative activity, 73%/500 a??g/mL and 37%/100 a??g/mL; protection activity, 69%/500 a??g/mL and 33%/100 a??g/mL), which is significantly higher than that of Ningnanmycin. The systematic study provides strong evidence that these simple thiourea derivatives could become potential TMV inhibitors.

Full text of DOI:10.1021/jf505355r

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Article
Application of "hydrogen bonding interaction" in new drug development:
Design, synthesis, antiviral activity and SARs of thiourea derivatives
Aidang Lu, Ziwen Wang, Zhenghong Zhou, Jianxin Chen, and Qingmin Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505355r • Publication Date (Web): 26 Jan 2015
Downloaded from http://pubs.acs.org on January 29, 2015
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Page 1 of 30
Journal of Agricultural and Food Chemistry
Application of “hydrogen bonding interaction” in new drug
development: Design, synthesis, antiviral activity and SARs of thiourea
derivatives
§,
Aidang Lu†,*, Ziwen Wang‡, *, Zhenghong Zhou§, Jianxin Chen†, Qingmin Wang§,*
†School of Marine Science and Engineering, Hebei University of Technology, Tianjin 300130, China
‡Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Key Laboratory of
InorganicꢀOrganic Hybrid Functional Material Chemistry, Ministry of Education, College of
Chemistry, Tianjin Normal University, Tianjin 300387, China;
§State Key Laboratory of ElementoꢀOrganic Chemistry, Research Institute of ElementoꢀOrganic
Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China
* To whom correspondence should be addressed. For Aidang Lu, Eꢀmail: luaidang@hebut.edu.cn;
Phone: 0086ꢀ22ꢀ60302241 Ext 205; Fax: 0086ꢀ22ꢀ60204274; For Ziwen Wang, Eꢀmail:
wangziwen2725@163.com; Phone: 0086ꢀ22ꢀ23503792; Fax: 0086ꢀ22ꢀ23503792; For Prof. Qingmin
Wang, Eꢀmail: wangqm@nankai.edu.cn; Phone: 0086ꢀ22ꢀ23503952; Fax: 0086ꢀ22ꢀ23503952.
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ABSTRACT: A series of simple thiourea derivatives were designed based on the
structure of nature product harmine and lead compound and synthesized from amines in
one step. The antiviral activity of these thiourea derivatives was evaluated. Most of them
exhibited significantly higher antiꢀTMV activity than commercial plant virucides
Ribavirin, harmine and lead compound. Hydrogen bond was found to be important but
not the more the better. The optimal compound (R,R)ꢀ20 showed the best antiꢀTMV
activity in vitro and in vivo (in vitro activity: 75%/500 ꢁg/mL and 39%/100 ꢁg/mL;
inactivation activity: 71%/500 ꢁg/mL and 35%/100 ꢁg/mL; curative activity: 73%/500
ꢁg/mL and 37%/100 ꢁg/mL; protection activity: 69%/500 ꢁg/mL and 33%/100 ꢁg/mL)
which is significantly higher than that of Ningnanmycin. The systematic study provides
strong evidence that these simple thiourea derivatives could become potential TMV
inhibitors.
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KEYWORDS: tobacco mosaic virus, nature product, thiourea derivatives, antiꢀTMV
activity, SARs
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INTRODUCTION
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Plant viruses have a great opportunity to infect their hosts due to their lower
immunity. It is extremely difficult to controlling virus diseases effectively. Tobacco
mosaic virus (TMV) may cause severe losses to agriculture, but its control is still a
challenge. So far, many plant viral inhibitors (BTH, Ningnanmycin, Ribavirin, etc) have
been used to combat TMV disease. However, all of chemical treatments can only reduce
the degree of plants’ infection.1
The agrochemicals derived from botanical have many advantages for its low
toxicity to humans. Another characteristic is their degradable ability which can reduce
their environmental risks. However, inevitably botanical agrochemicals also have some
disadvantages such as too complex to synthesize; active ingredients easy to decompose;
most botanical agrochemicals play a pharmacodynamics slowly, need to spray frequently
due to very short residual efficient duration, and so on. These shortcomings limit the
widespread use of botanical pesticides. Therefore, to discover new pesticide based on a
precursor from active substances of plant origin is an important and a feasible means to
solve the problem above.2
In our previous work, we found that nature product harmine (Figure 1) exhibited
excellent antiꢀTMV activity (antiviral activity against TMV in vitro and inactivation,
curative and protection activity in vivo were 45%, 41%, 39%, and 42%, respectively, at
500
ꢁg/mL).
Meanwhile,
(1S,3S)ꢀ1ꢀmethylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoleꢀ3ꢀcarbohydrazide (Figure 1,
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Lead compound) maintained high antiꢀTMV activity (the corresponding activity were
50%, 46%, 48%, and 50%, respectively, at 500 ꢁg/mL), and it exhibited excellent
stability and solubility; these advantages made it an ideal lead compound for further
derivation.3
Chiral thioureas and their derivatives possess many different catalytic and biological
activities. Part of them can not only support help to control configuration of compounds
as catalysts but also have antiꢀHIV activity as medicines.4–6 Some compounds bearing
thiourea against TMV have been reported by Jin,7 Li,8 Song,9 Yang,10 and Yuan.11
From theoretical and experimental points of view, much attention was focused on
intermolecular forces for their essential role in determining the threeꢀdimensional
structure. They are commonly seen in Proteins, DNA as well as enzymeꢀsubstrate
complexes.12 There are widespread reports about hydrogen bonding over the past ten
years. However, the applications of hydrogen bonding in drug design are only now
coming to light. Thiourea derivatives can bind to the nitrogen or oxygen atom serving as
catalysts.13 TMV, composing primarily with protein and RNA, contains a series of amino
acid residues.14 If they can bind to the residues of TMV, simple thiourea derivatives
would exhibit antiviral activity.
A series of simple thiourea derivatives were designed based on the structure of
nature product harmine and lead compound and synthesized as potential TMV inhibitors
(Figure 1). The structures of these thiourea derivatives were confirmed and assessment
for their antiꢀTMV was systematically evaluated.
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MATERIALS AND METHODS
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Instruments. The melting points of the products were determined on an Xꢀ4 binocular
microscope (Gongyi Yuhua Instrument Co., China) and the thermometer was not
corrected. NMR spectra were acquired with a Bruker 400 MHz (100 MHz for 13C)
instrument at room temperature. Chemical shifts were measured relative to residual
solvent peaks of CDCl3 (1H: δ = 7.26 ppm; 13C: δ = 77.0 ppm) or d6ꢀDMSO (1H: δ = 2.50
ppm; 13C: δ = 39.5 ppm) with tetramethylsilane as internal standards. The following
abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet,
t = triplet, m = multiplet, and bs = broad singlet. All firstꢀorder splitting patterns were
assigned on the basis of multiplet appearance. Splitting patterns that could not be easily
interpreted were designated multiplet (m) or broad (br). HRMS data were obtained with
an FTꢀICR MS spectrometer (Ionspec, 7.0 T). Analytical TLC was performed on silica
gel GF 254. Column chromatographic purification was performed using silica gel. All
reagents were of analytical reagent grade or chemically pure and purified prior to use
when necessary. Compounds 18–22 (Figure 2) were prepared according to our
previously reported procedure.15 The isothiocyanates were prepared according to the
literature.16
General Experimental Procedures
General Procedures for the Preparation of Compounds 1–8, 13–17 and 20.17
To a mixture of CH2Cl2 (10 mL) and amines (5.0 mmol) was added the
corresponding isothiocyanate (5.0 mmol or 10 mmol), and the resulting mixture was
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stirred at room temperature until TLC indicated the reaction was complete. The reaction
mixture was evaporated in vacuum. The pure products 1–8 and 13–17 were obtained by
recrystallization from petroleum ether and ether. Compounds 20 were purified or
chromatography on a column of silica gel with PEꢀEtOAc (v/v = 1: 1).
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1,3-Diphenylthiourea (1): white solid; yield, 99%; mp 144−145 °C (lit.18 140−142 °C);
13
90
1H NMR (400 MHz, CDCl3) δ 8.10 (s, 2H), 7.35−7.40 (m, 10H); C NMR (100 MHz,
91
CDCl3) δ 179.8, 137.2, 129.6, 127.1, 125.3; HRMS (ESI) calcd for C13H12N2S (M+H)+
229.0794, found 229.0791.
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1,3-Dibenzylthiourea (2): white solid; yield, 98%; mp 142−144 °C (lit.19 145−146 °C);
1H NMR (400 MHz, CDCl3) δ 7.28−7.34 (m, 6H), 7.02 (d, J = 6.8 Hz, 4H), 6.13 (br s,
2H), 4.61 (s, 4H); 13C NMR (100 MHz, CDCl3) δ 181.9, 136.6, 129.0, 128.0, 127.5, 48.6;
HRMS (ESI) calcd for C15H16N2S (M+H)+ 257.1107, found 257.1101.
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1,3-Dicyclohexylthiourea (3): white solid; yield, 99%; mp 176−177 °C (lit.18
174−176 °C); 1H NMR (400 MHz, CDCl3) δ 5.64 (br s, 2H), 3.84 (s, 2H), 1.99−2.02 (m,
4H), 1.60−1.73 (m, 6H), 1.32−1.41 (m, 4H), 1.13−1.24 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 179.0, 52.9, 32.9, 25.4, 24.7; HRMS (ESI) calcd for C13H24N2S (M+H)+
241.1733, found 241.1738.
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1-(3,5-Bis(trifluoromethyl)phenyl)-3-phenylthiourea (4): white solid; yield, 99%; mp
135−136 °C (lit.17 141−143 °C); 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.97 (s, 1H),
7.35 (s, 1H), 7.68 (s, 1H), 7.51 (t, J = 6.4 Hz, 2H), 7.40−7.42 (m, 1H), 7.32−7.34 (d, J =
6.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 179.8, 139.5, 135.5, 132.1 (JCꢀF = 33.6 Hz),
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130.6, 128.4, 125.6, 124.7 (JCꢀF = 2.5 Hz), 124.3 and 121.6 (JCꢀF = 271.1 Hz), 119.82 (m) ;
HRMS (ESI) calcd for C15H10F6N2S (M+H)+ 365.0542, found 365.0546.
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1-(3,5-Bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea (5): white solid; yield, 100%;
mp 159−160 °C (lit.17 164−165 °C); 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 7.76 (s,
2H), 6.13 (s, 1H), 4.20 (s, 1H), 2.04−2.08 (m, 2H), 1.61−1.72 (m, 3H), 1.36−1.45 (m,
2H), 1.17−1.22 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 178.9, 139.1, 132.7 (JCꢀF = 34.7
Hz), 126.8, 124.1 and 121.4 (JCꢀF = 271.4 Hz), 118.7, 53.9, 32.3, 25.2, 24.5; HRMS (ESI)
calcd for C15H16F6N2S (M+H)+ 371.1011, found 371.1017.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-butylthiourea (6): white solid; yield, 98%; mp
94−97 °C; 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 2H), 7.70 (s, 1H), 7.60 (s, 1H), 6.13 (br
s, 1H), 3.62 (s, 2H), 1.58−1.65 (m, 2H), 1.34 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 180.3, 139.1, 132.8 (JCꢀF = 33.6 Hz), 126.8, 124.1 and 121.4 (JCꢀF
=
271.3 Hz), 118.7, 45.1, 30.7, 20.1, 13.6; HRMS (ESI) calcd for C13H14F6N2S (M+H)+
345.0855, found 345.0852.
1-(3,5-Bis(trifluoromethyl)phenyl)-3-(tert-butyl)thiourea (7): white solid; yield, 98%;
1
mp 160 °C (dec.); H NMR (400 MHz, CDCl3) δ 8.44 (br s, 1H), 7.77 (s, 2H), 7.72 (s,
1H), 6.13 (s, 1H), 1.55 (s, 9H); 13C NMR (100 MHz, DMSOꢀd6) δ 179.7, 142.4, 130.5
(JCꢀF = 32.4 Hz), 127.8, 125.1 and 122.4 (JCꢀF = 271.1 Hz), 119.7, 53.6, 28.7; HRMS (ESI)
calcd for C13H14F6N2S (M+H)+ 345.0855, found 345.0853.
1,3-Bis(3,5-bis(trifluoromethyl)phenyl)thiourea (8): white solid; yield, 99%; mp
174−176 °C (lit.20 170−172 °C); 1H NMR (400 MHz, DMSOꢀd6) δ 10.65 (s, 2H), 8.21 (s,
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4H), 7.85 (s, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 180.5, 141.2, 130.3 (JCꢀF = 32.9 Hz),
124.4, 124.4 and 121.713 (JCꢀF = 271.1 Hz), 117.5; HRMS (ESI) calcd for C17H8F12N2S
(M+H)+ 501.0289, found 501.0293.
1,1'-((1R,2R)-1,2-Diphenylethane-1,2-diyl)bis(3-phenylthiourea) (13): white solid;
yield, 100%; mp 179−181 °C (lit.21 198 °C); [α]2D0
=
+
63.2 (c = 0.5, CHCl3); 1H NMR (400
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MHz, DMSOꢀd6) δ 9.75 (s, 2H), 8.43 (s, 2H), 7.10−7.34 (m, 20H), 5.98 (s, 2H); C
NMR (100 MHz, DMSOꢀd6) δ 180.9, 134.0, 139.6, 129.1, 128.6, 128.5, 127.7, 124.8,
123.5, 62.7; HRMS (ESI) calcd for C28H26N4S2 (M+H)+ 483.1672, found 483.1669.
1,1'-((1R,2R)-1,2-Diphenylethane-1,2-diyl)bis(3-benzylthiourea) (14): white solid;
yield, 100%; mp 195−196 °C; [α]2D0
= +
75.4 (c = 0.1, CHCl3); 1H NMR (400 MHz,
DMSOꢀd6) δ 8.10 (s, 4H), 7.08−7.27 (m, 20H), 5.87 (s, 2H), 4.72 (s, 2H), 4.57 (s, 2H);
13C NMR (100 MHz, DMSOꢀd6) δ 182.6, 139.9, 139.0, 128.2, 128.0, 127.7, 127.2, 127.1,
126.8, 62.0, 47.1; HRMS (ESI) calcd for C28H26N4S2 (M+H)+ 483.1672, found 483.1669.
1,1'-((1R,2R)-1,2-Diphenylethane-1,2-diyl)bis(3-benzhydrylthiourea) (15): white
solid; yield, 93%; mp 109−111 °C; [α]2D0
= +
84.2 (c = 0.5, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 7.06−7.39 (m, 30H), 6.75 (br s, 6H), 5.86 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 180.5, 140.5, 139.5, 137.7, 129.1, 128.4, 128.2, 128.0, 127.8, 127.7, 127.6, 127.1, 65.1,
62.2; HRMS (ESI) calcd for C42H38N4S2 (M+H)+ 663.2611, found 663.2609.
1,1'-((1R,2R)-1,2-Diphenylethane-1,2-diyl)bis(3-(3,5-bis(trifluoromethyl)phenyl)thio
urea) ((1R,2R)-16): white solid; yield, 92%; mp 192−194 °C (lit.22 194−196 °C); [α]2D0
−50.7 (c = 0.5, CHCl3); 1H NMR (400 MHz, DMSOꢀd6) δ 10.39 (s, 2H), 8.77 (s, 2H), 8.16
=
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(s, 4H), 7.69 (s, 2H), 7.22−7.38 (m, 10H), 5.97 (s, 2H); 13C NMR (100 MHz, DMSOꢀd6)
δ 180.2, 141.3, 138.4, 129.8−130.5 (m), 128.3, 128.1, 127.8, 127.6, 126.5, 124.3, 122.0,
121.5, 118.8, 116.0, 62.4; HRMS (ESI) calcd for C32H22F12N4S2 (M+H)+ 755.1167, found
755.1163.
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1,1'-((1R,2R)-cyclohexane-1,2-diyl)bis(3-(3,5-bis(trifluoromethyl)phenyl)thiourea)
((1R,2R)-17): white solid; yield, 95%; mp 132−134 °C (lit.23 132−133 °C); [α]2D0 = −61.2 (c
= 0.5, CHCl3); 1H NMR (400 MHz, DMSOꢀd6) δ 10.15 (s, 2H), 8.18 (s, 6H), 7.71 (s, 2H),
4.35 (s, 2H), 2.20 (s, 2H) , 1.72 (s, 2H) , 1.31 (s, 4H); 13C NMR (100 MHz, DMSOꢀd6) δ
180.0, 141.6, 130.1 (JCꢀF = 32.7 Hz), 127.2, 124.5 and 121.8 (JCꢀF = 271.0 Hz), 119.1,
56.8, 31.1, 24.2; HRMS (ESI) calcd for C24H20F12N4S2 (M+H)+ 657.1011, found
657.1016.
1-((1R,2R)-2-Amino-1,2-diphenylethyl)-3-benzylthiourea ((R,R)ꢀ20): white solid;
yield, 63%; mp 53−55 °C; [α]2D0
= +
69.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ
7.42 (br s, 1H), 7.12−7.30 (m, 15H), 6.52 (br s, 1H), 5.09 (br s, 1H), 4.59−4.62 (m, 1H),
13
4.51 (dd, J = 5.2 and 15.2 Hz, 1H), 4.29 (d, J = 4.0 Hz, 1H), 1.50 (br s, 2H); C NMR
(100 MHz, CDCl3) δ181.8, 141.9, 139.8, 137.2, 128.81, 128.76, 128.6, 127.67, 127.62,
127.4, 126.7, 126.4, 64.0, 60.2, 48.2; HRMS (ESI) calcd for C22H23N3S (M+H)+
362.1685, found 362.1684.
1-((1S,2S)-2-Amino-1,2-diphenylethyl)-3-benzylthiourea ((S,S)ꢀ20): white solid; yield,
65%; mp 52−54 °C; [α]2D0
= − = −74.3 (c = 1.0, CHCl3));
69.7 (c = 1.0, CHCl3) (lit.24 [α]2D4 1H
NMR (400 MHz, CDCl3) δ 7.47 (br s, 1H), 7.11−7.27 (m, 15H), 6.58 (br s, 1H), 5.12 (br
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s, 1H), 4.58−4.62 (m, 1H), 4.50 (dd, J = 4.8 and 14.8 Hz, 1H), 4.28 (d, J = 4.0 Hz, 1H),
1.66 (br s, 2H); 13C NMR (100 MHz, CDCl3) δ 181.8, 141.8, 139.7, 137.2, 128.82,
128.75, 128.6, 127.7, 127.6, 127.4, 126.7, 126.3, 63.9, 60.2, 48.3; HRMS (ESI) calcd for
C22H23N3S (M+H)+ 362.1685, found 362.1683.
General Procedures for the Preparation of Componds 9–12.25
In a 100 mL threeꢀnecks flask, the isothiocyanatobenzene (20 mmol), amino acids or
their derivatives (20 mmol), NaOH (22 mmol), water (20 mL) and THF (40 mL) were
added and stirred at room temperature for 24 h. After completion of the reaction, the pH
of the mixture was adjusted to pH 2ꢀ3 with aqueous 25% HCl, and then solvents were
removed under vacuum. The remaining residue was recrystallized from absolute
methanol to obtain the desired thiourea derivatives 9–12.
(S)-2-(3-Phenylthioureido)propanoic acid (9): white solid; yield, 75%; mp 173−175 °C;
[α]2D0 = −44.2 (c = 0.5, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.84 (br s, 1H), 7.46−7.53 (m,
13
3H), 7.32 (d, J = 8.0 Hz, 2H), 4.35 (q, J = 6.8 Hz, 1H), 1.59 (d, J = 6.8 Hz, 3H); C
NMR (100 MHz, CDCl3) δ 183.5, 174.1, 132.5, 129.3, 129.2, 128.2, 55.5, 17.0; HRMS
(ESI) calcd for C10H12N2O2S (M+H)+ 225.0692, found 225.0695.
(S)-3-Phenyl-2-(3-phenylthioureido)propanoic acid (10): white solid; yield, 78%; mp
182−184 °C (lit.25 184−185 °C); [α]2D0 = −43.2 (c = 0.5, CH3OH); 1H NMR (400 MHz,
CDCl3) δ 7.70 (s, 1H), 7.42−7.47 (m, 3H), 7.33−7.39 (m, 3H), 7.26 (d, J = 6.8 Hz, 3H),
7.05 (d, J = 6.8 Hz, 2H), 4.53 (dd, J = 4.0 and 7.6 Hz, 1H), 3.34 (dd, J = 4.0 and 14.0 Hz,
13
1H), 3.11 (dd, J = 7.6 and 14.0 Hz, 1H); C NMR (100 MHz, CDCl3) δ 183.7, 172.9,
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209
210
133.9, 132.4, 129.7, 129.4, 129.2, 128.2, 127.8, 60.9, 37.4; HRMS (ESI) calcd for
C16H16N2O2S (M+H)+ 301.1005, found 301.1004.
(S)-1-(3-Oxo-1-phenylpentan-2-yl)-3-phenylthiourea (11): white solid; yield, 65%; mp
152−154 °C; [α]2D0 = −55.0 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSOꢀd6) δ 8.79 (s, 1H),
7.27−7.39 (m, 10H), 6.68 (s, 1H), 4.06 (t, J = 6.4 Hz, 1H), 3.04−3.07 (m, 1H), 2.89−2.94
(m, 1H), 1.03−1.10 (m, 1H), 0.57 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ
180.8, 137.8, 137.4, 129.9, 129.4, 128.1, 128.0, 127.0, 126.2, 94.4, 61.2, 34.7, 29.6, 8.0;
HRMS (ESI) calcd for C18H20N2OS (M+H)+ 313.1369, found 313.1368.
(S)-1-(1-Oxo-1,3-Diphenylpropan-2-yl)-3-phenylthiourea (12): white solid; yield,
68%; mp 152−154 °C; [α]2D0 = −58.2 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSOꢀd6) δ
9.13 (s, 1H), 7.53 (s, 1H), 7.06−7.30 (m, 15H), 4.31 (t, J = 7.2 Hz, 1H), 2.95−3.10 (m,
13
2H); C NMR (100 MHz, DMSOꢀd6) δ 181.1, 140.7, 137.9, 137.1, 129.5, 129.1, 128.7,
127.8, 127.6, 127.4, 126.7, 126.4, 125.9, 94.7, 67.9, 33.8; HRMS (ESI) calcd for
C22H20N2OS (M+H)+ 361.1369, found 361.1367.
Antiviral Biological Assay. The procedure of purifying TMV and the method to test the
antiꢀTMV activity of the thiourea derivatives were the same with those reported
previously in the literature.26
Purification of Tobacco Mosaic Virus. The upper leaves of Nicotiana tabacum L.
inoculated with TMV were selected and ground in phosphate buffer and then filtered
through doubleꢀlayer pledget. The filtrate was centrifuged at 10000g, treated with PEG
twice, and centrifuged again. The whole experiment was processed at 4 °C. Absorbance
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value was estimated at 260 nm by ultraviolet spectrophotometer.
0.1%, 260nm
1cm
Virus concn = (A260 × dilution ratio)E /
Antiviral Activity of Compounds against TMV in Vitro. Fresh leaf of the 5ꢀ6 growth
stage of tobacco inoculated by the juiceꢀleaf rubbing method (concentration of TMV is
5.88 × 10ꢀ2 ꢀg/mL) was cut into halves along the main vein. The halves were immersed
into the solution of different concentrations (see Table 1) of the compounds and double
distilled water for 20min, respectively, and then cultured at 25 °C for 72 h. Every
concentration for each compound was replicated at least three times.
Protective Effect of Compounds against TMV in Vivo. The compound solution was
smeared on the left side and the solvent serving as control on the right side of growing N.
tabacum L. leaves of the same ages. The leaves were then inoculated with the virus after
12 h. A brush was dipped in TMV of 6 × 10ꢀ3 mg/mL to inoculate the leaves, which were
previously scattered with silicon carbide. The leaves were then washed with water and
rubbed softly along the nervature once or twice. The local lesion numbers appearing 3ꢀ4
days after inoculation were counted. There are three replicates for each compound.
Inactivation Effect of Compounds against TMV in Vivo. The virus was inhibited by
mixing with the compound solution at the same volume for 30min. The mixture was then
inoculated on the left side of the leaves of N. tabacum L., whereas the right side of the
leaves was inoculated with the mixture of solvent and the virus for control. The local
lesion numbers were recorded 3ꢀ4 days after inoculation. There are three replicates for
each compound.
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Curative Effect of Compounds against TMV in Vivo. Growing leaves of N. tabacum L.
of the same ages were selected. TMV (concentration of 6.0 × 10ꢀ3 mg/mL) was dipped
and inoculated on the whole leaves. Then the leaves were washed with water and dried.
The compound solution was smeared on the left side, and the solvent was smeared on the
right side for control. The local lesion numbers were then counted and recorded 3ꢀ4 days
after inoculation. There are three replicates for each compound. The in vitro and in vivo
inhibition rates of the compound were then calculated according to the following formula
(“av” means average, and controls were not treated with compound).
Inhibition rate (%) = [(av local lesion no. of control—av local lesion no. of
drugꢀtreated)/av local lesion no. of control] × 100%
RESULTS AND DISCUSSION
Chemistry.
Synthesis of the Compounds
procedures in Figure 3. Amines were reacted with corresponding isothiocyanate to give
thioureas 13 17 in almost quantitatively yields. Compounds 12 were obtained
1−17. Compounds 1−17 were synthesized according to
1
−8
,
−
9−
from isothiocyanatobenzene and amino acids or their derivatives in the presence of
NaOH in good yields.
Phytotoxic Activity. Compounds 1−22 were first tested for their phytotoxic activity
against the test plant.27 The results indicated that compounds
activity at 500 ꢁg/mL.
1−22 showed no phytotoxic
Antiviral Activity. The antiꢀTMV activity of compounds
1−22 was tested. The
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commercial plant virucide Ningnanmycin, perhaps the most successful registered
antiꢀplant viral agent, Ribavirin, nature product harmine and lead compound were used as
the controls.
In Vitro AntiꢁTMV Activity. The in vitro antiviral assay results of the synthesized
compounds were listed in Table 1. As the control, Ribavirin exhibited a 41% inhibitory
effect at 500 ꢁg/mL, whereas most of the synthesized compounds 1−22 exhibited higher
antiviral activity than Ribavirin even at the concentration of 100 ꢁg/mL. The optimal
compound 20 exhibited significantly higher inhibitory effect than Ningnanmycin,
harmine and lead compound emerging as a new lead compound for antiꢀTMV research.
To investigate the influence of the hydrogen bond strength on the antiꢀTMV activity,
compounds
the best inhibitory effect (inhibitory effect:
steric effect, thioureas
1
,
4
, and
8
were tested. Thiourea
8
with the stronger hydrogen bond did show
1 ≈ 4 For studying the influence of the
exhibited 45%, 56%, 32%,
8
>
).
4
−
7
were synthesized. Compounds
4
−7
and 45% inhibitory effect at 500 ꢁg/mL, respectively. Comparing the results of
antiꢀTMV activity was improved by increasing steric hindrance of the thiourea.
4−7, the
Compounds 9−12 were prepared from isothiocyanatobenzene and natural amino acids
or their derivatives. Compound 11 showed the best in vitro antiꢀTMV activity among
them with 44% inhibitory effect.
Bisꢀthioureaꢀtype derivatives 13−17 displayed in vitro activity ranging from
37%−56% against TMV at 500 ꢁg/mL. Amongst them, (R,R)ꢀ16 bearing 1,2ꢀ
diphenylethylenediamine skeleton showed higher activity than (R,R)ꢀ17 containing
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1,2ꢀcyclohexanediamine(46% and 42%, respectively). Compound 13 afforded good
result with inhibitory rate of 56%. All of primary amine thioureas 18
higher activity than Ribavirin, harmine and lead compound. Compounds 20 exhibited
best result with inhibitory rate of >70%. The optical pure compounds 16 18 and 20 were
−22 exhibited much
,
prepared to investigate the effect of configuration on antiviral activity.
(R,R)ꢀConfiguration is confirmed to be the optimal in vitro antiviral configuration
(inhibitory effect: (R,R)ꢀ16 > (±)ꢀ16 > (S,S)ꢀ16; (R,R)ꢀ18 > (±)ꢀ18 > (S,S)ꢀ18; (R,R)ꢀ20
(±)ꢀ20> (S,S)ꢀ20).
>
As shown in Table 1, most of the compounds 1−22 displayed good in vitro activity
against TMV. Therefore, these compounds were bioassayed further to investigate their
antiviral activity in vivo.
In Vivo AntiꢁTMV Activity. As shown in Table 2, most of the compounds also displayed
higher in vivo activity than that of the Ribavirin. (R,R)ꢀ20 showed the best result in vivo
antiꢀTMV activity (inactivation activity: 71%/500 ꢁg/mL and 35%/100 ꢁg/mL; curative
activity: 73%/500 ꢁg/mL and 37%/100 ꢁg/mL; and protection activity: 69%/500 ꢁg/mL
and 33%/100 ꢁg/mL), which is significantly higher than that of Ribavirin (inactivation
activity: 31%/500 ꢁg/mL and 12%/100 ꢁg/mL; curative activity: 39%/500 ꢁg/mL and
18%/100 ꢁg/mL; and protection activity: 47%/500 ꢁg/mL and 17%/100 ꢁg/mL) and
Ningnanmycin (inactivation activity: 63%/500 ꢁg/mL and 28%/100 ꢁg/mL; curative
activity: 54%/500 ꢁg/mL and 22%/100 ꢁg/mL; and protection activity: 64%/500 ꢁg/mL
and 26%/100 ꢁg/mL).
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Just as the antiviral activity in vitro, Compounds
vivo activity, and the results disclosed that the antiꢀTMV activity related to the hydrogen
bond strength and steric effect in molecules. Thiourea bearing
bisꢀ3,5ꢀBis(trifluoromethyl)phenyl gave relatively higher activity (60%, 56%, and 58% at
500 ꢁg/mL) than that of
Chiral thiourea derivatives
34%−49% against TMV at 500 ꢁg/mL, and different activity patterns displayed different
activity rule (inactivation activity: 11 10 12; curative activity: 11 10 12
protection activity: 11 10 12
The bisꢀthiourea derivatives showed relatively lower activity than corresponding
single thiourea derivatives (in vivo activity: 19 13 20 14 18 16) which indicated
that the hydrogen bond is important but not the more the better. All of primary amine
thioureas 18 22 exhibited much higher in vivo activity than Ribavirin, harmine and lead
compound, especially for compounds 20
1−8 displayed moderate to good in
8
1−7.
9−12 showed moderate antiviral activity ranging from
9
>
>
>
>
>
9
>
;
>
>
> 9).
>
;
>
;
>
−
.
In summary, based on the structure of nature product harmine and lead compound, a
series of simple thiourea derivatives were designed and synthesized from amines in one
step. The antiviral activity of these compounds were evaluated in vitro and in vivo. Most
of them exhibited significantly higher antiviral activity against TMV than commercial
plant virucide Ribavirin. The optimal compound (R,R)ꢀ20 showed excellent antiꢀTMV
activity in vitro and in vivo (in vitro activity: 75%/500 ꢁg/mL and 39%/100 ꢁg/mL;
inactivation activity: 71%/500 ꢁg/mL and 35%/100 ꢁg/mL; curative activity: 73%/500
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ꢁg/mL and 37%/100 ꢁg/mL; and protection activity: 69%/500 ꢁg/mL and 33%/100
ꢁg/mL) which is significantly higher than that of Ribavirin, Ningnanmycin, harmine and
lead compound. The hydrogen bond is favorable for antiviral activity within a certain
range but not the more the better. Present study provides fundamental support for
development and optimization of these simple thiourea derivatives as potential inhibitors
of plant virus. Further studies on structural optimization and mode of action are currently
underway in our laboratories.
ASSOCIATED CONTENT
Supporting Information
13
1H and C NMR spectra of compounds
1
−17 and 20. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(A.L.) Eꢀmail: luaidang@hebut.edu.cn; Phone: 0086ꢀ22ꢀ60302241 Ext 205; Fax:
0086ꢀ22ꢀ60204274
*(Z.W.) Eꢀmail: wangziwen2725@163.com; Phone: 0086ꢀ22ꢀ23503792; Fax:
0086ꢀ22ꢀ23503792.
*(Q.W.) Eꢀmail: wangqm@nankai.edu.cn. Phone: 0086ꢀ22ꢀ23503952. Fax:
0086ꢀ22ꢀ23503952
Funding
This study was supported by Natural Science Fund of China (21302038, 21402142,
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21132003), Natural Science Fund of Hebei Province (B2013202237), and PCSIRT
(IRT1059).
Notes
The authors declare no competing financial interest.
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Figure Captions
Figure 1. Design of Thiourea Derivatives
Figure 2. Chemical Structures of 18 22
Figure 3. Synthesis of Thiourea Derivatives
–
1–17
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Table 1. In Vitro Antiviral Activity of Compounds Ribavirin, Harmine, Ningnanmycin,
Lead Compound and 1−22 against TMV
Concn
(ꢁg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
Inhibition
rate (%)a
45±1
10±1
32±2
10±2
18±1
0
Concn
(ꢁg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
Inhibition
rate (%)a
36±1
8±2
Compd
compd
(S,S)-16
(±)-16
1
41±2
16±1
42±2
16±1
57±2
29±1
51±1
22±2
53±1
25±2
57±1
23±2
75±2
39±2
70±1
33±2
73±2
37±1
44±2
0
2
3
(R,R)-17
(R,R)-18
(S,S)-18
(±)-18
45±2
5±1
4
56±2
17±1
32±2
9±1
5
6
45±1
10±2
51±2
32±2
36±1
0
7
(±)-19
8
9
(R,R)-20
(S,S)-20
(±)-20
40±2
6±1
10
44±2
8±2
11
(R,R)-21
(R,R)-22
33±1
0
48±1
13±1
50±2
21±1
45±2
20±1
41±2
10±1
61±1
26±1
12
56±2
23±1
37±1
15±1
40±1
8±1
Lead
13
compound
Harmine
14
15
Ribavirin
46±2
18±2
(R,R)-16
Ningnanmycin
aAverage of three replicates; All results are expressed as mean ± SD.
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Table 2. In Vivo Antiviral Activity of Compounds Ribavirin, Harmine, Ningnanmycin,
Lead Compound and 1−22 against TMV
Concn
(ꢁg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
Inactivation
Curative
Protection
Concn
(ꢁg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
Inactivation
effect (%)a
40±1
19±1
46±2
20±1
49±2
16±2
57±1
24±2
55±1
19±2
56±2
22±2
61±2
27±1
71±2
35±1
66±2
29±1
69±2
31±1
44±1
19±2
45±1
14±2
46±2
17±1
41±1
11±1
31±2
12±1
63±2
28±1
Curative
Protection
Compd
Compd
(S,S)-16
(±)-16
effect (%)a effect (%)a effect (%)a
effect (%)a effect (%)a
50±1
18±3
36±1
9±1
42±2
13±1
43±3
18±2
24±1
0
40±1
15±1
40±1
18±1
27±2
0
46±3
20±1
48±1
21±1
47±2
18±2
55±1
29±2
50±1
25±2
53±2
26±2
63±1
32±2
73±1
37±2
70±1
30±2
70±1
35±1
48±1
18±2
50±1
19±2
48±2
20±2
39±1
16±2
39±2
18±2
54±1
22±1
43±1
13±1
49±2
19±1
50±1
19±1
60±1
27±2
53±2
21±2
58±1
26±2
61±2
26±1
69±2
33±1
64±1
31±2
68±2
33±1
48±1
20±1
48±2
22±1
50±1
23±1
42±2
16±1
47±1
17±1
64±2
26±1
1
2
17±2
0
3
(R,R)-17
(R,R)-18
(S,S)-18
(±)-18
49±1
18±1
59±1
20±1
38±2
15±1
43±2
18±1
60±1
25±2
49±1
12±1
46±2
24±1
48±2
13±1
41±2
0
41±2
12±1
53±3
28±1
43±1
10±1
41±2
11±1
56±1
22±2
41±1
17±1
47±2
23±1
49±1
20±2
36±1
9±1
42±1
15±1
50±1
24±1
35±2
9±1
4
5
6
39±1
16±1
58±1
28±1
34±2
11±1
42±1
21±1
46±2
15±1
37±2
14±1
60±1
24±1
40±1
10±2
50±2
20±1
54±1
20±1
7
(±)-19
8
9
(R,R)-20
(S,S)-20
(±)-20
10
11
(R,R)-21
(R,R)-22
12
58±1
26±1
33±1
0
61±2
30±1
30±3
0
Lead
13
compound
14
Harmine
Ribavirin
41±2
13±2
53±1
25±1
47±1
21±1
49±2
23±1
15
Ningnanmy
cin
(R,R)-16
a Average of three replicates; All results are expressed as mean ± SD.
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Journal of Agricultural and Food Chemistry
Figure 1.
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Figure 2.
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Journal of Agricultural and Food Chemistry
Figure 3.
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