Synthesis and antiviral bioactivity of chiral thioureas containing leucine and phosphonate moieties

Article abstract of DOI:10.3390/molecules15085112

A series of novel chiral thioureas 3a-n bearing leucine and phosphonate moieties were synthesized in excellent yields. The structures of the compounds were completely characterized by elemental analysis, IR, ¹H-, ¹³C-, ³¹P- and ¹⁹F-NMR spectral data. A half-leaf method was used to determine the in vivo protective and curative efficacies of the title products against tobacco mosaic virus (V). The compounds 3l and 3n displayed good in vivo protection and curative effects against V with inhibitory rates of 60.1, 62.8% (protection) and 56.7, 53.6% (curative) at 0.5 mg/mL, respectively. To the best of our knowledge, this is the first report on the antiviral activity of chiral thioureas containing leucine and phosphonate moieties.

Full text of DOI:10.3390/molecules15085112

Molecules 2010, 15, 5112-5123; doi:10.3390/molecules15085112
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antiviral Bioactivity of Chiral Thioureas
Containing Leucine and Phosphonate Moieties
Jingzi Liu 1,2, Song Yang 1,*, Xiangyang Li 1, Huitao Fan 1, Pinaki Bhadury 1, Weiming Xu 1, Jian
Wu 1 and Zhencao Wang 1
1
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, China; E-Mails: liujingz@126.com (J.L.);
michael.lee1983@yahoo.com.cn (X.L.); fanhuitao0818@163.com (H.F.); bhadury@gzu.edu.cn (P.B.);
xuweiming2009@163.com(W.X); jianwu2691@yahoo.com.cn(J.W); wzc.4884@163.com (Z.W)
2
Laboratory of Medicinal Chemistry, Zunyi Medical College, Zunyi, 563003, China
* Author to whom correspondence should be addressed; E-Mail: gdjhzx@gmail.com;
Tel.: +86 851 362 0521; Fax: +86 851 362 2211.
Received: 18 June 2010; in revised form: 26 July 2010 / Accepted: 27 July 2010 /
Published: 29 July 2010
Abstract: A series of novel chiral thioureas 3a-n bearing leucine and phosphonate
moieties were synthesized in excellent yields. The structures of the compounds were
13
31
19
completely characterized by elemental analysis, IR, 1H-, C-, P- and F-NMR spectral
data. A half-leaf method was used to determine the in vivo protective and curative
efficacies of the title products against tobacco mosaic virus (TMV). The compounds 3l and
3n displayed good in vivo protection and curative effects against TMV with inhibitory rates
of 60.1, 62.8% (protection) and 56.7, 53.6% (curative) at 0.5 mg/mL, respectively. To the
best of our knowledge, this is the first report on the antiviral activity of chiral thioureas
containing leucine and phosphonate moieties.
Keywords: chiral thiourea; leucine; phosphonate; antiviral activity; synthesis

Molecules 2010, 15
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1. Introduction
Chiral thioureas and their derivatives constitute an important class of compounds which exhibit a
wide range of antibacterial, fungicidal, herbicidal, antiviral and plant growth regulatory activities.
Some of them can serve not only as chiral organocatalysts in the synthesis of asymmetric compounds
[1], but also as potential anticancer and anti-HIV drugs [2-4]. Amongst the noteworthy contributions
reported in this field, Venkatachalam and his group [4] obtained chiral naphthyl thioureas (CNT) as
potent non-nucleoside inhibitors (NNI) of the reverse transcriptase (RT) enzyme of HIV-1.
Interestingly, the R-enantiomers of these derivatives could inhibit the recombinant RT in vitro with
lower IC50 values compared to their corresponding S-enantiomers. Unfortunately, chiral thiourea
derivatives have scarcely been evaluated for their plant antiviral activities for agricultural applications.
It is well known that certain bioisosteres of natural amino acids and a-aminophosphonic acid
analogues are associated with significant bioactivities and are often employed as enzyme inhibitors,
antimicrobial, antitumor and antiviral agents [5-9]. Further, suitably substituted phosphonic analogues
of L-leucine can play prominent role in leucine aminopeptidase inhibition and in the early stages of
HIV infection [10,11]. In this context, we have previously discovered some novel α-aminophosphonate
derivatives with appreciable antiviral activity against the tobacco mosaic virus (TMV) [12-14]. Based
on these findings, we turned our attention to the incorporation of the thiourea and α-aminophosphonate
pharmacophores into a single structure through a chiral L-leucine linker. The idea was to generate new
potentially antiviral chiral thiourea derivatives 3a-n with high activity, low toxicity and minimal
residues [15]. The synthetic route is shown in Scheme 1. The structures of the target compounds were
1
firmly established by IR, H-, C-, P- and F-NMR spectra and elemental analysis. Preliminary
13
31
19
bioassay tests showed that some of these compounds possessed good in vivo anti-TMV activities.
Scheme 1. Synthetic route to chiral thiourea derivatives 3a-n.
R2
NHBoc
R2
NHBoc
H
N
TFA, CH2Cl2, r.t.
Na2CO3, 0~5
HBTU, DIPEA
CH2Cl2, r.t.
OH
R1NH2
+
R1
O
O
1
S
R2
R2
NH2
R
H
N
H
N
O
P
OR
THF, r.t.
O
O
+
R1
N
H
N
H
R
R1
S=C=N
P
OR
O
O
O
2a-e
8a-d
3a-n
8a:R=Et;
3a:R1=C6H5CH2, R2=i-Bu, R=Et; 3b:R1=C6H5CH2, R2=i-Bu, R=n-Pr;
3c:R1=C6H5CH2, R2=i-Bu, R=i-Pr; 3d:R1=C6H5CH2, R2=i-Bu, R=n-Bu;
3e:R1=C6H5CH2, R2=t-Bu, R=Et; 3f:R1=C6H5CH2, R2=t-Bu, R=n-Pr;
3g:R1=C6H5CH2, R2=t-Bu, R=i-Pr; 3h:R1=C6H5CH2, R2=t-Bu, R=n-Bu;
3i:R1=o-FC6H4CH2, R2=i-Bu, R=Et; 3j:R1=o-FC6H4CH2, R2=i-Bu, R=i-Pr;
3k:R1=p-FC6H4C H2, R2=i-Bu, R=Et; 3l:R1=p-FC6H4CH2, R2=i-Bu, R=i-Pr;
3m:R1=p-CF3C6 H4, R2=i-Bu, R=Et; 3n:R1=p-CF3C6 H4, R2=i-Bu, R=i-Pr.
2a:R1=C6H5CH2, R2=i-Bu;
2b:R1=C6H5CH2, R2=t-Bu;
2c:R1=o-FC6H4CH2, R2=i-Bu;
2d:R1=p-FC6H4CH2, R2=i-Bu;
2e:R1=p-CF3C6H4, R2=i-Bu;
8b:R=n-Pr;
8c:R=i-Pr;
8d:R=n-Bu
2. Results and Discussion
2.1. Chemistry
The synthetic route designed for the chiral thiourea analogues 3a-n containing
L-leucine and an
α-aminophosphonate moiety is summarized in Scheme 1. Boc-protected intermediate amide 1 was first

Molecules 2010, 15
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generated from the reaction of
L-N-Boc-leucine and a substituted arylamine in the presence of O-
benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) at room temperature.
The α-amino carboxamide derivatives 2a-e were obtained through Boc deprotection using
trifluoroacetic acid, as shown in Scheme 1. The desired chiral thiourea analogues 3a-n were then
prepared by the addition of O,O′-dialkylisothiocyanato(phenyl)methylphosphonates 8a-d to 2a-e in
THF at room temperature. The intermediates 8 were in turn obtained from benzaldehyde in five steps
by following a literature procedure [13], as shown in Scheme 2.
Scheme 2. Synthetic route to intermediates 8a-d.
R
O
N (E)
(E)
O
R
R
O
N
(E)
N
CHO
O
O
P
R
HP
O
NH3·H2O
75 -78
4
5
O
S
R
R
R
R
R
O
O
P
O
P
O
Et3N, CS2
O
· H2O
O
O
O
O
O
NH3·H2O
OH
P
R
O
Cl3COCOCCl3
NH3· OTs
NH2
N=C=S
7a-d
8a-d
6
7a:R=Et;
8a:R=Et;
7b:R=n-Pr;
7c:R=i-Pr;
7d:R=n-Bu
8b:R=n-Pr;
8c:R=i-Pr;
8d:R=n-Bu
The existing method for the conversion of product 7 into 8 by the use of phosphorus oxychloride
(POCl3) was however not satisfactory. This step of the reaction was further optimized by employing
different reagents, such as bis(trichloromethyl) carbonate (BTC), thiophosgene (CSCl2) and 1,1-
thiocarbonyldiimidazole (TCDI). The results of these experiments were compared with those obtained
with the phosphorus oxychloride method (Table 1). As could be observed from the data, the best result
was obtained with BTC, which afforded 67-79% isolated yield of 8a-8d against the reported yield [13]
of 54-62%. Therefore, BTC was selected as the ideal reagent for this key conversion.
Table 1. Comparison of the yield for intermediates 8a-d using different reagents.
Yield (%)
Compd.
R
CSCl2
TCDI
BTC
POCla
Et
24
trace
79
71
67
75
60
62
54
58
8a
8b
8c
8d
n-Pr
i-Pr
n-Bu
a Reported yields in the reference [13].
With regard to the best solvent that could be used for the preparation of title compounds 3 from 2
and 8 (Scheme 1), the model compound 3e was synthesized in different solvents, e.g. tetrahydrofuran
(THF), acetonitrile (CH3CN), acetone (CH3COCH3), dichloromethane (CH2Cl2) and toluene and the
results are provided in Table 2. Under optimized conditions, a maximum yield of 98% could be
obtained when the reaction mixture was stirred at room temperature in the solvent THF for 0.5 h.

Molecules 2010, 15
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Table 2. Role of different solvents for the synthesis of 3e.
No.
1
Solvent
THF
Vol. (mL)
Reaction time (h)
Temperature (ºC)
Yield (%)
5
5
5
5
5
0.5
0.5
0.5
0.5
0.5
room
room
room
room
room
98
76
89
73
CH3CN
CH3COCH3
CH2Cl2
2
3
4
toluene
55.7
5
All the products were unequivocally characterized by IR and NMR spectral data and elemental
analyses. The characteristic IR absorption bands at 3269-3467 cm-1, 1645-1678 cm-1, 1546-1564 cm-1,
1212-1227 cm-1, and 1008-1028 cm-1 confirmed the presence of NH, C=O, C=S, P=O and P-O-C
1
functional groups, respectively. In the H-NMR spectra of title compounds 3a-n, all aromatic protons
displyed the expected multiplet near 6.78-7.79 ppm. While the amidic NH protons appeared downfield
in the 7.74-10.12 ppm range as broad singlets, the NH protons of thioureido groups revealed weak
peaks due to the existence of hydrogen bonds. The typical phosphorus resonance at 19.7-22.8 ppm in
31
the P-NMR spectra of all target compounds confirmed the presence of a phosphorus center coupled
19
to an adjacent CH. In the F-NMR spectra, the fluorine resonance due to Ar-F and CF3 appeared
at -115.1 to -118.8 ppm and -63.2 ppm, respectively. The typical carbon resonances at 183.1-184.6 and
13
170.6-174.4 ppm in the C-NMR spectra of the title compounds were indicative of the existence of
C=S and C=O double bonds, respectively. All the nonequivalent carbon atoms were identified in the
1
13C-NMR and the total number of protons calculated from the H-NMR integration curves was in
complete agreement with the assigned structures.
2.2. Antiviral activity
The results of the in vivo bioassay against TMV are given in Table 3. Ningnanmycin was used as
the reference antiviral agent. Among the studied compounds, thiourea derivatives 3l
(R1= p-FC6H4CH2, R2= i-Bu, R= i-Pr) and 3n (R1= p-CF3C6H4, R2= i-Bu, R= i-Pr) displayed
remarkable protection activities (60.1 and 62.8%, respectively) and curative rates (56.7 and 53.6%,
respectively) against TMV, which were comparable to the protection activity (53.1%) and curative rate
(55.3%) shown by the commercial reference ningnanmycin. The results indicated that the antiviral
activity depended to a certain extent on the type of stereochemical configuration and the nature of the
substituents present in the compound. In particular, the title compounds derived from iso-leucine
(R2= i-Bu) showed better anti-TMV activity compared to those derived from tert-leucine
(R2= tert-Bu). Thus, the following trend in curative trend was noted: 3a (42.3%) > 3e (28.4%), 3b
(35.4%) > 3f (33.5%), 3c (46.2%) > 3g (40.9%) and 3d (31.2%) > 3h (24.1%). Furthermore, iso-
leucine-derived thioureas incorporating O,O’-diisopropylphosphonate (R= i-Pr) were found to be
superior in terms of anti-TMV activity compared to their phosphonate analogues which were obtained
from diethyl, di-n-propyl or di-n-butyl phosphites (R= Et, n-Pr, n-Bu). Evidently, a phosphonate
moiety derived from branched alkyl chains should be the preferred over straight chain alkylated ones
in the design of the title thioureas. In addition, the compounds bearing an electron withdrawing group
(R1= p-F, p-CF3) at the 4-position of the aromatic ring showed better anti-TMV activities, with
inhibition rates ranging from 48.1 to 56.7%. At for the absolute configuration, it was observed that the
enantiomer D-3l and racemate ( )-3l did not reveal any noticeable anti-TMV activity (curative rates

Molecules 2010, 15
5116
28.4% and 34.9%, respectively). This was in sharp contrast to
L
-3l (derived from
L-leucine) which
showed significant activity against TMV. In order to establish a definite structure-activity relationship,
some structural modification needs to be conducted by making subtle changes in the nature of
substituents of title thioureas having
L-configuration.
Table 3. The protection effect and curative effect of the compounds 3a-n against TMV in vivo.
Concentration
Protection
Effect (%)
19.6*
27.5*
52.8**
12.3*
34.0*
45.1*
23.4*
9.8*
Curative
Effect (%)
42.3*
Compd.
R1
R2
R
(mg/mL)
0.5
0.5
C6H5CH2
C6H5CH2
C6H5CH2
i-Bu
i-Bu
Et
n-Pr
i-Pr
n-Bu
Et
3a
3b
35.4*
46.2*
i-Bu
i-Bu
0.5
0.5
3c
3d
C6H5CH2
C6H5CH2
31.2*
28.4*
tert-Bu
tert-Bu
tert-Bu
tert-Bu
i-Bu
0.5
0.5
3e
C6H5CH2
C6H5CH2
C6H5CH2
n-Pr
i-Pr
n-Bu
Et
33.5*
40.9*
24.1*
45.3*
3f
3g
3h
3i
0.5
0.5
o-FC6H4CH2
o-FC6H4CH2
p-FC6H4CH2
p-FC6H4CH2
p-FC6H4CH2
p-FC6H4CH2
p-CF3C6H4
p-CF3C6H4
–
0.5
0.5
43.4*
48.3**
44.9*
60.1**
–
i-Bu
i-Bu
i-Pr
Et
51.3**
48.1**
56.7**
28.4*
3j
3k
0.5
0.5
0.5
0.5
i-Bu
i-Bu
i-Pr
i-Pr
i-Pr
Et
3l
D-3l
i-Bu
i-Bu
–
34.9*
3l
0.5
0.5
51.2*
62.8**
53.1**
49.8**
53.6*
55.3*
3m
3n
i-Bu
–
i-Pr
–
0.5
Ningnanmycin
n = 3 for all groups; * P < 0.05, ** P < 0.01.
3. Experimental
3.1. General
The melting points of the products were determined on a XT-4 binocular microscope (Beijing Tech
Instrument Co., China) and were not corrected. The IR spectra were recorded on a Bruker VECTOR22
13
31
spectrometer in KBr disks. The 1H, C,19 F and P NMR spectra (solvent DMSO-d6 or CDCl3) were
recorded at room temperature on a JEOL-ECX 500 NMR spectrometer operating at 500, 125, 470 and
200 MHz, respectively, using TMS as an internal standard. Data are reported as follows: chemical
shifts in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad and
m = multiplet, coupling constant (Hz) and integration. Elemental analyses were performed on an
Elementar Vario-III CHN analyzer. The reagents were all of analytical grade or chemically pure.
Analytical TLC was performed on silica gel GF254 plates.
3.2. Preparation of intermediates 2a-e
L-N-Boc-leucine (2.31 g, 0.01 mol) and O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexa-
fluorophosphate (HBTU, 3.80 g, 0.01 mol) were loaded into an oven-dried round bottomed flask
equipped with a magnetic stir bar, rubber septum, and argon inlet. Anhydrous dichloromethane
(50 mL) was then added. After 3 minutes, anhydrous DIPEA (2.58 g, 0.02 mol) and arylamine
(0.011 mol) were sequentially added and the reaction mixture was stirred at room temperature for
2-4 h. During the process, the state of the solution was seen to change from slightly heterogeneous to

Molecules 2010, 15
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homogeneous confirming the consumption of HBTU. The reaction mixture was poured into a
separatory funnel containing 1N HCl (50 mL), and was then partitioned between dichloromethane and
aqueous hydrochloric acid solution. The organic layer was washed with 1N HCl (3 × 30 mL), dried
over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting light yellow oil was
transferred to a 100-mL flask and redissolved in dichloromethane (30 mL). Trifluoroacetic acid (7 mL)
was then added in one portion. After 2-4 h, the mixture was slowly partitioned with dichloromethane
and chilled, saturated aqueous sodium carbonate solution (50 mL) was added. The aqueous layer was
extracted with dichloromethane (3 × 30 mL), and the combined organic extracts were dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by thin
layer chromatography (TLC) on a silica gel (developing solvent: 3-4% MeOH/CH2Cl2, V/V) to give the
intermediates 2a-e.
1
-2-Amino-N-benzyl-4-methylpentanamide (2a). White crystals, yield 77%, m.p. 60-62 ºC; H-NMR
L
(CDC13): δ 0.95 (d, 6H, J = 3.2 Hz, 2CH3), 1.73-1.75 (m, 2H, CH2), 1.96 (br s, 2H, NH2), 2.80-2.82
(m, 1H, CH), 3.45-3.48 (m, H, CH), 4.43 (d, J = 6.3 Hz, 2H, NCH2), 7.26-7.35 (m, 5H, ArH), 7.72 (br
13
s, 1H, NH); C-NMR (CDC13): δ 175.6, 138.6, 128.7, 127.8, 127.4, 53.6, 44.1, 43.2, 24.9, 21.4; IR
(KBr, cm-1): v 3304, 2958, 2868, 1647; Anal. Calcd. for C13H20N2O: C 70.87, H 9.15, N 12.72; Found:
C 70.62, H 9.34, N 12.86.
1
-2-Aamino-N-benzyl-3,3-dimethylbutanamide (2b). White crystals, yield 83%, m.p. 53-54 ºC, H-
L
NMR (CDC13, 500 MHz): δ 0.99 (s, 9H, 3CH3), 1.56 (br s, 2H, NH2), 3.11 (s, 1H, CH), 4.42 (d,
J = 3.45 Hz, 2H, NCH2), 7.25-7.31 (m, 5H, ArH), 7.78 (br s, 1H, NH); 13C-NMR (CDC13, 125 MHz):
δ 173.6, 138.6, 128.7, 128.0, 127.5, 64.5, 43.2, 34.3, 26.9; IR (KBr, cm-1): v 3306, 2946, 1650; Anal.
Calcd. for C13H20N2O: C 70.87, H 9.15, N 12.72; Found: C 70.71, H 9.23, N 12.36.
L-N-(2-Fluorobenzyl)-2-amino-4-methylpentanamide (2c). Colorless viscous liquid, yield 68%;
1H-NMR (CDC13): δ 0.99 (d, 6H, J = 4.6 Hz, 2CH3), 1.23 (t, 2H, J = 7.2 Hz, CH2), 1.65 (br s, 2H,
NH2), 1.76-1.84 (m, 1H, CH), 3.52-3.55 (m, H, CH), 4.23 (d, J = 5.3 Hz, 2H, NCH2), 7.05-7.33(m, 4H,
ArH), 8.43 (br s, 1H, NH); 13C-NMR (CDC13): δ 174.2, 159.7, 131.4, 128.8, 115.3, 124.1, 53.2, 43.5,
34.6, 24.6, 21.3; 19F-NMR (CDC13): δ -115.5; IR (KBr, cm-1) v: 3308, 2960, 2873, 1649; Anal. Calcd.
for C13H19FN2O: C 65.52, H 8.04, N 11.76; Found: C 65.36, H 8.32, N 11.65.
L
-N-(4-Fluorobenzyl)-2-amino-4-methylpentanamide (2d). Colorless liquid, yield 73%, nD25 1.5055;
1H-NMR (CDC13): δ 0.90 (d, 6H, J = 3.4 Hz, 2CH3), 1.63-1.72 (m, 2H, CH2), 2.25 (br s, 2H, NH2),
2.76-2.79 (m, 1H, CH), 3.35-3.38 (m, 2H, CH), 4.34 (d, J = 5.15 Hz, 2H, NCH2), 6.96-7.22 (m, 4H,
13
ArH), 7.98 (br s, 1H, NH); C-NMR (CDC13): δ 174.7, 161.5, 137.4, 128.6, 115.2, 52.9, 44.5, 42.8,
24.7, 21.4; 19F-NMR (CDC13): δ -115.3; IR (KBr, cm-1): v 3309, 2954, 2870, 1645, 1504, 1223; Anal.
Calcd. for C13H19FN2O: C 65.52, H 8.04, N 11.76; Found: C 65.33, H 8.26, N 11.51.
L
-2-Amino-4-methyl-N-(4-(trifluoromethyl)phenyl)pentanamide (2e). Colorless liquid, yield 56%,
1
n25 1.4750; H-NMR (CDC13): δ 0.95 (d, 6H, J = 6.3 Hz, 2CH3), 1.43-1.47 (m, 1H, CH), 1.75-1.79
D
(m, 1H, CH2), 2.51 (br s, 2H, NH2), 3.53 (d, 1H, J = 4.0 Hz, , NCH), 7.54-7.78(d, 4H, J = 8.6 Hz,
ArH), 9.99 (br s, 1H, NH); 13C-NMR (CDC13): δ 174.7, 141.3, 125.9, 125.6, 123.3, 119.1, 54.0, 43.7,

Molecules 2010, 15
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24.8, 21.4; F-NMR (CDC13): δ -62.0; IR (KBr, cm-1): v 3462, 3269, 2953, 1628, 1506, 1323, 1115,
19
1067; Anal. Calcd. for C13H17F3N2O: C 56.93, H 6.25, N 10.21; Found: C 57.18, H 6.47, N 10.39.
3.3. Preparation of O,O′-dialkylisothiocyanato(phenyl)methylphosphonate intermediates 8a-d
To a solution of α-aminophosphonate (6 mmol) in ether (15 mL), triethylamine (18 mmol) was
added with constant stirring at room temperature and cooled to 0 ºC. Then, carbon disulfide (6 mmol)
was added dropwise and the mixture stirred for 1 h at 0 ºC, the temperature was raised to 23 ºC, and
stirring was continued for an additional 2 h. BTC (2 mmol) dissolved in ether (10 mL) was then added
dropwise into the reaction mixture and stirred for 3 h at 23 ºC. The solid was filtered off, and the liquid
was extracted with ether, treated with saturated sodium bicarbonate, and dried on anhydrous sodium
sulfate, filtered. Removal of the solvent followed by chromatography of the crude product on silica
using a mixture of petroleum ether and ethyl acetate as the developing solvent gave the intermediates 8
in 67-79% yields; data for 8a-d can be found in reference [13].
3.4. Preparation of title chiral thioureas 3a-n
A solution of O, O’-dialkylisothiocyanato(phenyl)methylphosphonate 8 (1 mmol) in
tetrahydrofuran (5 mL) was stirred, followed by dropwise addition of chiral amine 2a-e (1.1 mmol).
The stirring was continued for 0.5 h at 23 ºC, the solvent was evaporated and the crude product was
purified by preparative TLC using a mixture of ethyl acetate and n-hexane (V:V = 1:1) as developing
solvent to give title compound 3a-n.
Diethyl (3-(
L-1-benzylamino-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methylphosphonate (3a).
1
White solid, yield 94%; m.p. 43-45 ºC; [
α
]20 -17.9º (c 0.1, acetone); H-NMR (CDC13, 500 MHz): δ
D
0.79 (d, 6H, J = 6.3 Hz, 2CH3), 1.06 (t, 6H, J = 5.75 Hz, 2CH3), 1.24-1.46(m, 2H, CH2), 1.65-1.72 (m,
1H, CH), 3.68-4.07 (m, 2H, NCH2), 4.12-4.49 (m, 4H, 2OCH2) , 5.07 (d, 1H, J = 6.7 Hz, CH), 6.28 (s,
13
1H, PCH), 6.45 (s, 1H, NH), 6.95-7.48 (m, 10H, ArH), 7.91 (s, 1H, NH), 8.82 (br s, 1H, NH); C-
NMR (CDCl3, 125 MHz): δ 183.1, 172.5, 139.2, 136.1, 128.5, 127.6, 126.9, 64.3, 58.1, 54.3, 44.5,
41.4, 26.4, 23.7, 16.3; 31P- NMR (CDCl3, 200 MHz): δ 21.8; IR (KBr, cm-1): v 3284, 3065, 2938, 1659,
1560, 1216, 1012; Anal. Calcd for C25H36N3O4PS: C 59.39, H 7.18, N 8.31; Found C 59.57, H 7.33,
N 8.15.
Dipropyl (3-(
L-1-benzylamino-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methylphosphonate (3b).
1
Colorless viscous liquid, yield 89%; [
α
]20 -15.8º (c 0.1, acetone); H-NMR (CDC13): δ 0.81-1.04 (m,
D
12H, 4CH3), 1.27-1.45 (m, 6H, 3CH2), 1.67-1.70 (m, 1H, CH), 3.71-4.09 (m, 2H, NCH2), 4.12-4.53
(m, 4H, 2OCH2), 5.10 (d, 1H, J = 6.4 Hz, CH), 6.31 (s, 1H, PCH), 6.48 (s, 1H, NH), 6.94-7.49(m,
13
10H, ArH), 7.95 (s, 1H, NH), 9.07 (br s, 1H, NH); C-NMR (CDC13): δ 183.5, 172.3, 138.4, 135.6,
31
128.8, 127.1, 126.7, 68.2, 62.5, 54.4, 43.8, 41.3, 26.5, 24.2, 22.7, 10.1; P-NMR (CDCl3): δ 21.5; IR
(KBr, cm-1): 3276, 3081, 2927, 1663, 1564, 1219, 1009 cm-1; Anal. Calcd for C27H40N3O4PS: C 60.77,
H 7.55, N 7.87; Found: C 60.52, H 7.81, N 7.69.
Diisopropyl (3-(
L-1-benzylamino-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methylphosphonate
1
(3c). White solid, yield 95%; m.p. 51-53 ºC; [
α
]20 -14.2º (c 0.1, acetone); H-NMR (CDC13): δ 0.82
D

Molecules 2010, 15
5119
(d, 18H, J = 6.3 Hz, 6CH3), 1.42-1.59 (m, 2H, CH2), 1.76-1.89 (m, H, CH), 4.26-4.48 (m, 2H, NCH2),
4.69-4.78 (m, 2H, 2OCH), 5.09 (d, 1H, J = 5.75 Hz, CH), 6.27 (s, 1H, PCH), 7.06-7.46 (m, 10H, ArH),
7.81 (s, 1H, NH), 8.73 (br s, 1H, NH); 13C-NMR (CDC13): δ 183.9, 172.0, 138.2, 135.5, 128.7, 128.5,
127.6, 127.2, 73.2, 56.4, 55.9, 43.5, 40.2, 38.7, 24.3, 23.1; 31P-NMR (CDCl3): δ 22.8; IR (KBr, cm-1): v
3285, 3063, 2929, 1674, 1558, 1215, 1014; Anal. Calcd for C27H40N3O4PS: C 60.77, H 7.55, N 7.87;
Found: C 60.63, H 7.24, N 7.66.
Dibutyl (3-(
L-1-benzylamino-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methylphosphonate (3d).
1
Colorless viscous liquid, yield 86%; [
α
]20 -16.4º (c 0.1, acetone); H-NMR (CDC13): δ 0.80-0.95 (m,
D
12H, 4CH3), 1.20-1.43 (m, 10H, 5CH2), 1.55-1.72 (m, 1H, CH), 3.66-4.05 (m, 2H, NCH2), 4.11-4.55
(m, 4H, 2OCH2), 5.04 (d, 1H, J = 6.3 Hz, CH), 6.27 (s, 1H, PCH), 6.43(s, 1H, NH), 6.92-7.44 (m,
13
10H, ArH), 7.83 (s, 1H, NH), 8.70 (br s, 1H, NH); C-NMR (CDC13): δ 184.5, 170.6, 138.2, 135.6,
31
128.9, 128.6, 127.8, 67.7, 65.8, 55.5, 43.4, 34.8, 32.2, 26.9, 18.8, 13.7; P-NMR (CDCl3): δ 22.1; IR
(KBr, cm-1): v 3269, 3082, 2953, 1670, 1550, 1220, 1024; Anal. Calcd for C29H44N3O4PS: C, 62.01; H,
7.90; N, 7.48; Found: C 62.28, H 7.67, N 7.72.
Diethyl (3-(
L-1-benzylamino-3,3-dimethyl-1-oxobutan-2-yl)thioureido)(phenyl)methylphosphonate (3e).
1
White crystals, yield 98%; m.p. 157-159 ºC; [
α
]20 +15.5º (c 0.1, acetone); H-NMR (CDC13): δ 0.98
D
(s, 9H, C(CH3)3), 1.07 (t, 6H, J = 6.9 Hz, 2CH3), 3.75-4.06 (m, 2H, NCH2), 4.10-4.46 (m, 4H, 2OCH2),
4.93 (d, 1H, J = 8.6 Hz, CH(t-Bu)), 6.26 (s, 1H, PCH), 6.47 (s, 1H, NH), 7.15-7.47 (m, 10H, ArH),
13
7.92 (s, 1H, NH), 9.03 (br s, 1H, NH); C-NMR (CDCl3): δ 184.3, 170.7, 138.2, 135.4, 128.7, 128.0,
31
127.4, 66.3, 64.1, 55.4, 43.4, 34.8, 26.9; 16.5; P-NMR (CDCl3): δ 21.7; IR (KBr, cm-1): v 3292,
3068, 2965, 2867, 1669, 1562, 1221, 1013; Anal. Calcd for C25H36N3O4PS: C 59.39, H 7.18, N 8.31;
Found: C 59.52, H 7.34, N 8.12.
Dipropyl
(3-(L-1-benzylamino-3,3-dimethyl-1-oxobutan-2-yl)thioureido)(phenyl)methylphosphonate
1
(3f). White crystals, yield 93%; m.p. 171-172 ºC; [
α
]20 +9.6º (c 0.1, acetone); H-NMR (CDC13): δ
D
0.76 (t, 6H, J = 6.85 Hz, 2CH3), 1.03 (s, 9H, 3CH3), 1.42-1.74 (m, 4H, 2CH2), 3.70-3.95 (m, 2H,
NCH2), 4.09-4.47 (m, 4H, 2OCH2), 4.94 (d, 1H, J = 8.6 Hz, CH(t-Bu)), 6.25 (s, 1H, PCH), 6.53 (s, 1H,
13
NH), 7.12-7.47 (m, 10H, ArH), 7.94 (s, 1H, NH), 9.11 (br s, 1H, NH); C-NMR (CDC13): δ 184.4,
170.6, 138.0, 135.2, 128.7, 128.4, 127.5, 69.4, 65.8, 55.3, 43.3, 34.7, 26.8, 24.0, 9.9; 31P-NMR
(CDCl3): δ 21.5; IR (KBr, cm-1): v 3288, 3056, 2952, 2873, 1677, 1543, 1218, 1012; Anal. Calcd for
C27H40N3O4PS: C 60.77, H 7.55, N 7.87; Found: C 60.52, H 7.71, N 7.59.
Diisopropyl
(3-(L-1-benzylamino-3,3-dimethyl-1-oxobutan-2-yl)thioureido)(phenyl)methylphos-
1
phonate (3g). White crystals, yield 96%; m.p. 176-177 ºC; [
α
]2D0 +7.2º (c 0.1, acetone); H-NMR
(CDC13, 500 MHz): δ 1.01 (s, 9H, 3CH3), 1.19 (d, 12H, J = 6.3 Hz, 4CH3), 4.22-4.46 (m, 2H, NCH2),
4.63-4.84 (m, 2H, 2OCH), 4.96 (d, 1H, J = 7.45 Hz, CH(t-Bu)), 6.32 (s, 1H, PCH), 6.38 (s, 1H, NH),
13
7.11-7.52 (m, 10H, ArH), 7.80 (s, 1H, NH), 9.05 (br s, 1H, NH); C-NMR (CDC13, 125 MHz): δ
184.6, 170.7, 138.3, 135.8, 128.9, 128.6, 127.4, 73.1, 66.4, 55.9, 43.2, 34.6, 27.0, 24.3; 31P-
NMR(CDCl3, 200 MHz): δ 19.7; IR (KBr, cm-1): v 3286, 3057, 2963, 2861, 1672, 1548, 1224, 1011;
Anal. Calcd for C27H40N3O4PS: C 60.77, H 7.55, N 7.87; Found: C 60.91, H 7.34, N 7.48.

Molecules 2010, 15
5120
Dibutyl
(3-(L-1-benzylamino-3,3-dimethyl-1-oxobutan-2-yl)thioureido)(phenyl)methylphosphonate
1
(3h). White crystals, yield 91%; m.p. 122-124 ºC; [
α
]20 +12.1º (c 0.1, acetone); H-NMR (CDC13): δ
D
0.79 (t, 6H, J = 7.45 Hz, 2CH3), 0.96 (s, 9H, 3CH3), 1.16-1.71 (m, 8H, 2 CH2CH2), 3.71-4.01 (m, 2H,
NCH2), 4.06-4.51 (m, 4H, 2OCH2), 4.92 (d, 1H, J = 8.6 Hz, CH(t-Bu)), 6.25 (s, 1H, PCH), 6.46 (s, 1H,
13
NH), 7.13-7.46 (m, 10H, ArH), 7.92 (s, 1H, NH), 9.04 (br s, 1H, NH); C-NMR (CDC13): δ 184.5,
31
170.6, 138.2, 135.6, 128.9, 128.6, 127.8, 67.7, 65.8, 55.5, 43.4, 34.8, 32.2, 26.9, 18.8, 13.7; P-NMR
(CDCl3): δ 21.5; IR (KBr, cm-1): v 3283, 3065, 2954, 1671, 1552, 1216, 1013; Anal. Calcd for
C29H44N3O4PS: C, 62.01; H, 7.90; N, 7.48; Found: C 62.24, H 7.63, N 7.69.
Diethyl
(3-(
L
-1-(2-fluorobenzylamino)-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methyl-
]2D0 -22.3º (c 0.1, acetone); 1H-NMR (CDCl3):
phosphonate (3i). Colorless viscous liquid, yield 92%; [
α
δ 0.85 (d, 6H, J = 6.8 Hz, 2CH3), 1.11 (t, 6H, J = 5.7 Hz, 2CH3), 1.31-1.53(m, 2H, CH2), 1.67-1.74 (m,
1H, CH), 3.67-3.96 (m, 2H, NCH2), 4.26-4.47 (m, 4H, 2OCH2) , 5.20 (d, 1H, J = 6.7 Hz, CH), 6.29 (s,
1H, PCH), 6.32 (s, 1H, NH), 6.79-7.45 (m, 9H, ArH), 7.86 (s, 1H, NH), 8.57 (br s, 1H, NH); 13C-NMR
(CDCl3): δ 183.5, 171.4, 159.8, 136.3, 131.3, 128.6, 127.2, 126.5, 124.1, 115.2, 63.9, 58.2, 54.4, 41.6,
34.1, 26.1, 23.3, 16.4; 31P-NMR (CDCl3): δ 22.7; 19F-NMR (CDCl3): δ -118.6; IR (KBr, cm-1): v 3290,
3085, 2977, 1662, 1557, 1216, 1025; Anal. Calcd for C25H35FN3O4PS: C 57.35, H 6.74, N 8.03; Found:
C 57.53, H 6.55, N 8.28.
Diisopropyl
(3-(
L
-1-(2-fluorobenzylamino)-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methyl-
]2D0 -17.2º (c 0.1, acetone); 1H-NMR (CDCl3):
phosphonate (3j). Colorless viscous liquid, yield 88%; [
α
δ 0.87 (d, 18H, J = 6.4 Hz, 6CH3), 1.36-1.43 (m, 2H, CH2), 1.51-1.62 (m, H, CH), 3.75-4.16 (m, 2H,
NCH2), 4.53-4.71 (m, 2H, 2OCH), 5.23 (d, 1H, J = 6.8 Hz, CH), 6.27 (s, 1H, PCH), 6.30 (s, 1H, NH),
13
6.82-7.43 (m, 9H, ArH), 7.74 (s, 1H, NH), 8.42 (br s, 1H, NH); C-NMR (CDCl3): δ 183.7, 171.2,
159.5, 136.4, 131.5, 128.8, 127.0, 126.7, 124.2, 115.3, 73.0, 59.1, 55.4, 41.4, 34.3, 26.6, 23.5, 16.3;
19
31P-NMR (CDCl3): δ 20.3; F-NMR (CDCl3): δ -118.8; IR (KBr, cm-1): v 3275, 3067, 2961, 1668,
1554, 1227, 1015; Anal. Calcd for C27H39FN3O4PS: C 58.78, H 7.13, N 7.62; Found: C 58.53, H 7.32,
N 7.34.
Diethyl
(3-(
L
-1-(4-fluorobenzylamino)-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methylphos-
]2D0 -16.7º (c 0.1, acetone); 1H-NMR (CDCl3):
phonate (3k). White solid, yield 94%; m.p. 31-32 ºC; [
α
δ 0.82 (d, 6H, J = 6.5 Hz, 2CH3), 1.09 (t, 6H, J = 5.8Hz, 2CH3), 1.27-1.52(m, 2H, CH2), 1.66-1.71 (m,
1H, CH), 3.71-3.99 (m, 2H, NCH2), 4.14-4.43 (m, 4H, 2OCH2) , 5.20 (d, 1H, J = 6.7 Hz, CH), 6.29 (s,
1H, PCH), 6.37 (s, 1H, NH), 6.74-7.45 (m, 9H, ArH), 8.01 (s, 1H, NH), 8.74 (br s, 1H, NH); 13C-NMR
(CDCl3): δ 183.6, 171.7, 160.9, 137.1, 135.8, 128.7, 127.1, 126.6, 115.4, 64.1, 58.5, 54.2, 44.2, 41.3,
26.3, 23.6, 16.5; 31P-NMR (CDCl3): δ 22.2; 19F-NMR (CDCl3): δ -115.1 ppm; IR (KBr, cm-1): v 3285,
3080, 2963, 1676, 1551, 1223, 1020 cm-1; Anal. Calcd for C25H35FN3O4PS: C 57.35, H 6.74, N 8.03;
Found: C 57.59, H 6.48, N 7.96
Diisopropyl
phosphonate (3l). White solid, yield 90%; m.p. 43-44 ºC; [
(CDCl3): δ 0.84 (d, 18H, J = 6.3 Hz, 6CH3), 1.39-1.57 (m, 2H, CH2), 1.64-1.69 (m, H, CH), 4.21-4.43
(3-(L-1-(4-fluorobenzylamino)-4-methyl-1-oxopentan-2-yl)thioureido)(phenyl)methyl-
1
α
]2D0 -13.3º (c 0.1, acetone); H-NMR

Molecules 2010, 15
5121
(m, 2H, NCH2), 4.63-4.72 (m, 2H, 2OCH), 5.19 (d, 1H, J = 5.8 Hz, CH), 6.25 (s, 1H, PCH), 6.35 (s,
1H, NH), 6.95-7.40 (m, 9H, ArH), 7.98 (s, 1H, NH), 8.71 (br s, 1H, NH); 13C-NMR (CDC13): δ 183.7,
172.1, 161.2, 137.3, 135.6, 128.8, 127.4, 126.8, 115.3, 73.3, 59.6, 55.7, 43.4, 40.8, 26.4, 24.5, 23.2;
19
31P-NMR (CDCl3): δ 21.9; F-NMR (CDCl3): δ -115.4; IR (KBr, cm-1): v 3287, 3072, 2968, 1678,
1548, 1220, 1008; Anal. Calcd for C27H39FN3O4PS: C 58.78, H 7.13, N 7.62; Found: C 58.64, H 7.35,
N 7.41.
Diethyl
(3-(L-4-methyl-1-oxo-1-(4-(trifluoromethyl)phenylamino)pentan-2-yl)thioureido)(phenyl)-
1
methylphosphonate (3m). Colorless viscous liquid, yield 84%; [α
]2D0 -8.9º (c 0.1, acetone); H-NMR
(CDCl3): δ 0.94 (d, 6H, J = 5.7 Hz, 2CH3), 1.13 (t, 6H, J = 6.9 Hz, 2CH3), 0.99-1.02(m, H, CH),
1.66-1.72 (m, 2H, CH2), 3.92-4.13 (m, 4H, 2OCH2), 5.03-5.11 (m, 1H, CH), 6.31 (s, 1H, PCH),
13
7.31-7.79 (m, 9H, ArH), 10.07 (s, 1H, NH); C-NMR (CDCl3): δ 183.8, 174.2, 141.7, 136.9, 128.4,
31
127.0, 126.5, 126.1, 125.7, 123.3, 119.8, 73.4, 58.2, 54.1, 41.4, 26.2, 23.4, 21.8; P NMR (CDCl3): δ
19
22.3; F-NMR (CDCl3): δ -63.4; IR (KBr, cm-1): v 3467, 3280, 2968, 1648, 1552, 1218, 1026; Anal.
Calcd for C25H33F3N3O4PS: C 53.66, H 5.94, N 7.51; Found: C 53.74, H 6.17, N 7.22.
Diisopropyl(3-(L-4-methyl-1-oxo-1-(4-(trifluoromethyl)phenylamino)pentan-2-yl)thioureido) (phenyl -
1
methylphosphonate (3n). Colorless viscous liquid, yield 79%; [α
]2D0 -10.5º (c 0.1, acetone); H-NMR
(CDCl3): δ 0.98 (d, 18H, J = 6.4 Hz, 6CH3), 1.36-1.42 (m, 1H, CH), 1.63-1.68 (m, H, CH2), 4.17-4.43
(m, 2H, 2OCH), 5.06-5.16 (m, 1H, CH), 6.33 (s, 1H, PCH), 7.29-7.74 (m, 9H, ArH), 10.12 (br s, 1H,
NH); 13C-NMR (CDC13): δ 183.9, 174.4, 141.5, 137.2, 128.5, 127.1, 126.8, 126.2, 125.8, 123.5, 119.4,
73.6, 58.5, 54.2, 41.3, 26.1, 23.2, 21.7; 31P-NMR (CDCl3): δ 22.5; 19F-NMR (CDCl3): δ -63.2; IR (KBr,
cm-1): v 3465, 3276, 2962, 1645, 1546, 1212, 1028; Anal. Calcd for C27H37F3N3O4PS: C 55.19, H 6.35,
N 7.15; Found: C 55.26, H 6.71, N 6.97.
3.5. Antiviral Biological Assays
3.5.1. Purification of TMV
Using Gooding’s method [16], the upper leaves of Nicotiana tabacum L inoculated with TMV were
selected and were grounded in phosphate buffer, then filtered through double layer pledget. The filtrate
was centrifuged at 10,000 g, treated twice with PEG, and centrifuged again. The whole experiment was
processed at 4 ºC. Absorbance value was estimated at 260 nm by ultraviolet spectrophotometer:
0.1%, 260nm
Virus concn = (A260 × dilution ratio)E /
1cm
3.5.2. Protection effect of compound against TMV in vivo
The compound solution was smeared on the left side while solvent served as the control on the right
side of Nicotiana tabacum. L leaves of the same ages. The leaves were then inoculated by the virus
after 12 h. Tobacco mosaic virus of 6 × 10-3 mg/mL was dipped with brush to inoculate on the leaves
which were previously scattered with silicon carbide. Then the leaves were washed with water and
rubbed softly along the nervure once or twice. The local lesion numbers appeared 3-4 days after
inoculation [17]. Triplicate repetitions were conducted for each compound.

Molecules 2010, 15
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3.5.3. Curative effect of compound against TMV in vivo
The leaves on Nicotiana tabacum. L of the same ages were selected. The tobacco mosaic virus with
6 ×10-3 mg/mL concentration 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 recorded 3-4 days after
inoculation [17]. For each compound, three repetitions were conducted to ensure the reliability of the
results:
av local lesion numbers of control (not treated with compoud)-av local lesion numbers smeared with drugs
Inhibition rate (%) =
av local lesion nubbers without drugs
4. Conclusions
A series of novel chiral thioureas containing α-aminophosphonate moieties were synthesized in
high yield under mild conditions by the addition in THF of O, O′-dialkyl isothiocyanato(phenyl)-
methylphosphonates 8 to L-leucine derivatives. The bioassay results showed that title compounds
exhibited moderate to good anti-TMV bioactivity. In particular, compounds 3l (R1= p-FC6H4CH2,
R2= i-Bu, R= i-Pr) and 3n (R1= p-CF3C6H4, R2= i-Bu, R= i-Pr) showed better biological activity than
other structurally related analogues. The L-leucyl and phosphonate moiety derived from branched alkyl
chains are preferred over straight chain alkylated ones in the design of the title thioureas, moreover, the
compounds bearing an electron withdrawing group (R1= p-F, p-CF3) at the 4-position of the aromatic
ring showed better anti-TMV activities. At for the absolute configuration, compound
from -leucine) showed more significant activity against TMV than its enantiomer
racemate ( )-3l. The present work demonstrates that the antiviral activity of chiral thioureas was
significantly improved through introduction of suitably substituted derivatives with -configuration.
L-3l (derived
L
D-3l and the
L
The influence of different substituents, minor structural modifications and steric parameters on
structure activity relationships for identifying lead bioactive compounds will be studied in our
future investigations.
Acknowledgements
The authors wish to thank the National Key Project for Basic Research (2010CB126105) and the
National Natural Science Foundation of China (20872021) for the financial support.
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Sample Availability: Samples of the compounds are available from the authors.
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