Synthesis, crystal structures, and in silico toxicity prediction of thienopyridine phosphoramidates

Article abstract of DOI:10.1080/00397911.2013.786092

New thieno[2,3-b]pyridine phosphoramidates compounds were synthesized and characterized by infrared; ¹H, ¹³C, and ³¹P NMR spectroscopy; and high-resolution mass spectrometry. The products were obtained in good yields (64-82%) under mild conditions by nucleophilic aromatic substitution reaction of aminoalkylphosphoramidates over 4-chlorothieno[2,3-b] pyridine-5-carbonitrile. The crystal structures of two compounds were solved by X-ray diffraction and showed a network of intermolecular interactions involving phosphoramidate groups. Druglike properties and toxicity of the new compounds were studied with the help of the software Molinspiration, Osiris, and Toxtree, and were compared with the standard drugs amphotericin B, miltefosine, benznidazole, and nifurtimox. [Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications for the following free supplemental resource(s): Full experimental and spectral details.]

Full text of DOI:10.1080/00397911.2013.786092

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Synthesis, Crystal Structures, and
in Silico Toxicity Prediction of
Thienopyridine Phosphoramidates
Leandro F. Pedrosa a , William P. de Macedo a , Antonia C. R. Furtado
a , Guilherme P. Guedes a b , Luiz C. S. Pinheiro a , Jackson A. L. C.
Resende a , Maria G. F. Vaz a , Alice M. R. Bernardino a & Marcos C.
de Souza a
a Instituto de Química, Universidade Federal Fluminense , Niterói ,
Brazil
b Departamento de Química , Instituto de Ciências Exatas,
Universidade Federal Rural do Rio de Janeiro , Seropédica , Brazil
Published online: 05 Sep 2013.
To cite this article: Leandro F. Pedrosa , William P. de Macedo , Antonia C. R. Furtado , Guilherme
P. Guedes , Luiz C. S. Pinheiro , Jackson A. L. C. Resende , Maria G. F. Vaz , Alice M. R. Bernardino
& Marcos C. de Souza (2013) Synthesis, Crystal Structures, and in Silico Toxicity Prediction of
Thienopyridine Phosphoramidates, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 43:24, 3373-3386
To link to this article: http://dx.doi.org/10.1080/00397911.2013.786092
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Synthetic Communications1, 43: 3373–3386, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2013.786092
SYNTHESIS, CRYSTAL STRUCTURES, AND IN SILICO
TOXICITY PREDICTION OF THIENOPYRIDINE
PHOSPHORAMIDATES
Leandro F. Pedrosa,1 William P. de Macedo,1
Antonia C. R. Furtado,1 Guilherme P. Guedes,1,2
Luiz C. S. Pinheiro,1 Jackson A. L. C. Resende,1
Maria G. F. Vaz,1 Alice M. R. Bernardino,1 and
Marcos C. de Souza1
1
´
Instituto de Quımica, Universidade Federal Fluminense, Niteroi, Brazil
Departamento de Quımica, Instituto de Ciencias Exatas, Universidade
´
2
´
ˆ
´
Federal Rural do Rio de Janeiro, Seropedica, Brazil
GRAPHICAL ABSTRACT
Abstract New thieno[2,3-b]pyridine phosphoramidates compounds were synthesized and
1
characterized by infrared; H, 13C, and 31P NMR spectroscopy; and high-resolution mass
spectrometry. The products were obtained in good yields (64–82%) under mild conditions
by nucleophilic aromatic substitution reaction of aminoalkylphosphoramidates over
4-chlorothieno[2,3-b]pyridine-5-carbonitrile. The crystal structures of two compounds were
solved by x-ray diffraction and showed a network of intermolecular interactions involving
phosphoramidate groups. Druglike properties and toxicity of the new compounds were stud-
ied with the help of the software Molinspiration, Osiris, and Toxtree, and were compared
with the standard drugs amphotericin B, miltefosine, benznidazole, and nifurtimox.
[Supplementary materials are available for this article. Go to the publisher’s online
edition of Synthetic Communications1 for the following free supplemental resource(s):
Full experimental and spectral details.]
Keywords Crystal structures; druglike properties; phosphoramidates; thienopyridine
Received February 21, 2013.
Address correspondence to Marcos C. de Souza, Instituto de Quımica, Universidade Federal
´
´
Fluminense, Niteroi, RJ 24020-141, Brazil. E-mail: gqomarc@vm.uff.br
3373

3374
L. F. PEDROSA ET AL.
INTRODUCTION
Fused heterocycles containing nitrogen and sulfur have been associated with
several biological and medicinal activities.[1] In particular, thienopyridine derivatives
play important roles because of their extensive spectrum of pharmacological
activities. They can be used as sedatives;[2] analgesics and anti-inflammatories;[3–5]
antiparasitic,[6] antibacterial,[7–10] and antitumor agents;[11–13] and antiviral
drugs[14–16] and in the treatment of diabetes mellitus,[17,18] antiplatelet aggre-
gation,[19–21] and myeloproliferative disorders.[22] Several 4-substituted thienopyri-
dines have been obtained by our group by nucleophilic substitution of the
4-chloro precursor with variable nucleophiles.
Besides, it is well known that phosphoramidates and their derivatives are
an important class of biologically relevant compounds.[23] Introduction of a
phosphoramidate group essentially changes the physical and chemical properties of
the parent molecule, accentuating the polarization and intermolecular bonding
characteristics.[24,25] The P O group plays a significant role as a strong hydrogen
=
bond acceptor, which is essential for the noncovalent bonding of proteins or other
specific ligands to their substrates.[26] The incorporation of a phosphoramidate group
into different heterocyclic systems constitutes an interesting synthetic strategy used in
the discovery of new drugs.[27] For example, it is widely used in association with
neutral lipophilic groups to form a lipophilic-membrane-permeable derivative able
to access intracellular target sites.[28] In the past few years, we focused our efforts
on the preparation of bioactive heterocycles containing phosphoramidate
groups.[29,30] In this work we report the synthesis of three new thieno[2,3-b]pyridine
phosphoramidate derivatives as well as the crystal structure of two of them. These
derivatives were submitted to in silico oral biodisponibility screening, drug score,
and druglikeness evaluation to analyze their overall potential to be qualified as drugs,
and also compared them to some antiparasitics for leishmaniasis and Chagas disease,
such as amphotericin B, miltefosine, benznidazole, and nifurtimox.
RESULTS AND DISCUSSION
Syntheses and Spectroscopic Measurements
The syntheses of the new thieno[2,3-b]pyridine phosphoramidates conjugates
(9a–c) were performed by nucleophilic aromatic substitution of the chlorine atom
in 4-substituted thieno[2,3-b]pyridine (5) by aminoalkylphosphoramidates 6a–c
(Scheme 1).
The starting 4-chlorothieno[2,3-b]pyridine-5-carbonitrile 5 was prepared from
2-nitrothiophene 1, which was reduced with tin and hydrochloric acid, leading to
the bis-(2-thienylammonium)hexachlorostannate 2. This compound was immediately
condensed with ethyl (ethoxymethylene)cyanoacetate in pyridine at 40–50 ꢀC over a
period of 24 h to produce ethyl a-cyano-b-(N-2-thienylammonium)acrylate 3 in
88% yield. The cyclization of the acrylate was carried out by refluxing in Dowtherm
at 250 ꢀC for 40 min, after which 4-hydroxythieno[2,3-b]pyridine-5-carbonitrile 4
was isolated in 78% yield, through precipitation from petroleum ether. Compound
4 was easily chlorinated refluxing phosphorus oxychloride at 110 ꢀC over a period
of 6 h to afford 4-chlorothieno[2,3-b]pyridine-5-carbonitrile 5 in 76% yield.[6,15,31]

THIENOPYRIDINE PHOSPHORAMIDATES
3375
Scheme 1. Reagents and conditions: (i) Sn, 35% HCl, 45 ꢀC; (ii) ethyl (ethoxymethylene)cyanoacetate,
pyridine, 40–50 ꢀC, 24 h; (iii) Dowtherm, 250 ꢀC, 40 min; (iv) POCl3, 110 ꢀC, 6 h; (v) CCl4, ethanol,
T < 55 ꢀC, 10 min; and (vi) THF, reflux, 9–12 h.
The aminoalkylphosphoramidates 6a–c were synthesized in 53–58% yield
from diisopropylphosphonate 7 and aliphatic diamines 8a–c.[29,32,33] To guarantee
monophosphorylation of the diamines, at least a 2.5-fold excess of diamine in
ethanol was used to maintain the (alkaline) pH necessary to catalyze the reaction.
Careful addition of 7 in ethanol and CCl4 over the diamine solution should not
exceed 10 min. At this stage, the temperature must be kept below 55 ꢀC, otherwise
bis-phosphorylation will occur preferentially. The nucleophilic aromatic substitution
of the chlorine atom in 4-substituted thieno[2,3-b]pyridine by amines has been used
as a versatile route to new thienopyridine derivatives.[16,34–36] The reaction of
4-chlorothieno[2,3-b]pyridine-5-carbonitrile 5 with an excess (2 equiv.) of aminoalkyl
phosphoramidates 6a–c in refluxing tetrahydrofuran (THF) for 9 to 12 h afforded
the thieno[2,3-b]pyridine phosphoramidate 9a–c in 64–82% yield. The presence of
an electron-withdrawing group (-CN) in the 5-position of the substrate 5 and the
excess of the nucleophilic agent 6a–c facilitates the nucleophilic substitution. The
1
products were fully characterized by infrared; H, 13C, and 31P NMR spectroscopy;
and high-resolution mass spectrometry (HRMS).
1
The H NMR spectra of compounds 9a–c compounds showed two doublets
3
around 7.58–8.04 and 7.30–7.46 ppm with J(HH) ꢁ 6.0 Hz corresponding to the
resonances of the thieno ring protons. The same spectra showed a singlet in the range
of 8.33–8.45 ppm attributable to the pyridine ring proton. The resonances of the iso-
propyl protons appeared as two doublets at 1.36–1.42 ppm and a doublet of septets
around 4.6 ppm with 3J(HH) ꢁ 6.1 Hz and 3J(PH) ꢁ 7.3 Hz. The NH signal was
detected as a broad singlet in the range 8.06–8.84 ppm. On the other hand, NHP pro-
tons showed coupling with phosphorus and the neighbor methylene group, giving rise
to a doublet of a triplet around 3.20 ppm with 3J(HH) ꢁ 7.0 Hz and 2J(PH) ꢁ 10.0 Hz.
In the aliphatic region, the assignment of the signals for methylene protons was based
on the correlation spectra (COSY) of these compounds. Typically, the methyne car-
2
bon signal in b position to phosphorus appears as a doublet with J(PC) ꢁ 5.2 Hz
around 71.5 ppm in 13C NMR spectroscopy. In all cases phosphorus and carbon

3376
L. F. PEDROSA ET AL.
showed coupling with 3J(PC) ꢁ 5.2 Hz, but no coupling 2J(PC) was observed.
The thieno[2,3-b]pyridine phosphoramidates 9a–c showed in their decoupled
31P NMR spectra one signal in the region of 7.78–9.84 pmm, typical for phosphora-
midates.[29,30,37,38] Furthermore, infrared spectra exhibited strong absorptions for the
ꢂ1
P O at 1194 cm , P-O around 1000–988 cmꢂ1, and weak absorption for the cyano
=
group at 2208 cmꢂ1
.
X-Ray Crystallography Investigation
Single crystals of 9a and 9b compounds suitable for x-ray diffraction were
obtained by slow solvent evaporation at room temperature. The crystal data and
structure refinement parameters for thieno[2,3-b]pyridine phosphoramidates 9a
and 9b are provided. The asymmetric units are shown in Fig. 1.
In these compounds, the phosphoramidate groups are linked to an aliphatic
skeleton through the C1 atom. The bond lengths and angles are typical of phosphor-
amidate groups (see Supplementary Information Tables S2 and S3). The
5-cyanothieno[2,3-b]pyridin-4-ylamino group is bonded to the alkyl chain through
C2 atom in 9a and C3 atom in 9b. It is important to highlight that the aromatic sub-
stituent group gives rise to weak intermolecular interactions and plays an important
role in the crystal packing. In compound 9a, intramolecular hydrogen bonding
involving N2 and O1 atoms lead to a seven-member ring, in which N1 and N2 atoms
are in an eclipsed conformation, in relation to the C1–C2 bond axis. A network of
hydrogen bonding in the [100] direction is observed due to the interaction between
N1 and O1 atoms (Fig. 2).
The bond distances and angles for hydrogen bonding are summarized in
Table 1. Besides, the molecular arrangement is also stabilized by weak interactions
between the pyridinic and thiophene rings from neighboring molecules.
As a result of these intra- and intermolecular interactions, the compound 9a
presents a more compact crystal packing, leading to a higher calculated density when
compared with 9b. The increase of one carbon atom in the alkyl spacer chain results
in a different crystal packing in 9b. The C1 and N2 atoms are in an eclipsed
conformation, viewed through the C2–C3 bond axis. This conformation does not
Figure 1. X-ray crystal structure of compounds 9a and 9b. Ellipsoids at 50% of probability.

THIENOPYRIDINE PHOSPHORAMIDATES
3377
Figure 2. Hydrogen bonding in compounds 9a and 9b. (Figure is provided in color online.)
allow intramolecular hydrogen bonding between N2 and O1 atoms, as observed in 9a.
Bifurcated intermolecular hydrogen bonding among O1, N1, and N2 are able to form
dimers of molecules (Fig. 2). Moreover, short contact C8-H8A ꢃꢃꢃ N2A in the [010]
direction and p ꢃꢃꢃ p stacking between the 5-cyanothieno[2,3-b]pyridine groups
stabilize the crystal packing. A search in the CCDC database shows a tendency of
phosphoramidates compounds to form dimers in the solid state, because 47% of
the compounds present in this base display this molecular arrangement.
This characteristic is associated to the torsion angle O1–P1–N1–C1, which is
close to 180ꢀ. In the compound 9a the intramolecular hydrogen bonding does not
allow the dimer formation, while in the 9b the longer alkyl chain permits a better
arrangement of the p ꢃꢃꢃ p stacking together with hydrogen bonds, leading to a supra-
molecular network.
Druglike Properties and Toxicity Prediction
In silico techniques for the druglike properties and prediction of toxicological
endpoints are extremely appealing because of their expeditious return of results
and their low cost. Moreover, these techniques can be used in a very early phase
of drug discovery even before the molecule is synthesized.[39] Free web-based
programs for Lipinski’s rule of five and toxicity prediction are available, including
the Molinspiration, Osiris, and Toxtree.
ꢀ
˚
Table 1. Hydrogen-bond geometry (A,
)
Compound
D—H ꢃꢃꢃ A
D—H
H ꢃꢃꢃ A
D ꢃꢃꢃ A
D—H ꢃꢃꢃ A
9a
N1—H1 ꢃꢃꢃ O1a
N2—H2 ꢃꢃꢃ O1
N1—H1 ꢃꢃꢃ O1b
N2—H2 ꢃꢃꢃ O1b
0.86
0.86
0.86
0.86
2.04
2.31
2.13
2.18
2.879 (3)
3.131 (3)
2.963 (3)
2.953 (3)
165
160
163
149
9b
ax ꢂ 1, y, z.
b–x þ 1, ꢂy þ 1, ꢂz þ 2.

3378
L. F. PEDROSA ET AL.
Molinspiration software was utilized to screen the compounds 9a–c based
on Lipinski’s rule of five and oral bioavailability and compared with standard
drugs against leishmaniasis and Chagas disease currently available (i.e., amphoteri-
cin B, miltefosine, benznidazole, and nifurtimox). The prediction results are in
Table 2.
According to stated procedures,[40–42] the parameters of Lipinski’s rule of five
are as follows: the molecular weight must be less than 500 Da, logP less than 5, the
amount of hydrogen donors (nOHNH) must be less than 5, and the amount of
acceptor hydrogen (nON) must be less than 10. miLogP (octanol=water partition
coefficient) is calculated by the methodology developed by Molinspiration as a sum
of fragment-based contributions and correction factors. The method is very robust
and is able to process practically all organic and most organometallic molecules.
For all the compounds the calculated logP values were around 2.058–2.6, below
the limit for the drugs to be able to penetrate through biomembranes according to
Lipinski’s rule. Thus, compounds 9a–c are expected to exhibit good bioavailability.
It is worth mentioning that all the compounds showed zero violation of the rule of
five, having successfully passed Lipinski’s rules. Two or more violations suggest the
probability of problems in bioavailability.
The oral bioavailability of drugs could be measured by the molecular weight,
number of rotatable bonds (nrotb), number of hydrogen bonds (nON and nOHNH),
and total polar surface area (TPSA). This set of criteria is called Veber’s rule.[43]
The oral bioavailability was marked by small molecular weight (less than 500);
also, the number of rotatable bond must be less than 10, the number of hydrogen
bond donors and acceptors must be less than 12, and TPSA values less than
˚
140 A. TPSA has been shown to be a very good descriptor characterizing drug
absorption, including intestinal absorption, bioavailability, and blood–brain barrier
penetration. Lipophilicity and polar surface area values are two important properties
for the prediction of oral bioavailability of drug molecules.[44] Molecules with TPSA
˚
values around 160 A or more are expected to exhibit poor intestinal absorption.
Table 2 shows that 9a–c are within this limit and in accordance with Veber’s rule.
Table 2. Molinspiration calculation of properties for Lipinski’s rulea
Acceptable range
miLog P
TPSA
natoms
MW
nON nOHNH nrotb nviolations
Compound
<5
<500
<10
<5
9a
9b
2.058
2.329
2.6
96.277
96.277
96.277
299.381
58.596
92.748
108.708
25.0
26.0
27.0
65.0
30.0
19.0
19.0
382.426
396.453
410.48
7
7
2
2
9
10
11
3
0
0
0
3
0
0
0
9c
7
2
Amphotericin B
Miltefosine
Benznidazole
Nifurtimox
0.365
1.303
0.778
0.709
922.119
449.657
260.253
287.297
17
5
12
0
23
5
7
1
8
0
3
amiLog P, Molinspiration-predicted log P; TPSA, total polar surface area; natoms, number of atoms;
MW, molecular weight; nON, number of hydrogen-bond acceptors; nOHNH, number of hydrogen-bond
donors; nrotb, number of rotatable bonds; and nviol, number of violations.

THIENOPYRIDINE PHOSPHORAMIDATES
3379
It has to be kept in mind that log P and TPSA values are the most important two
features, although not sufficient criteria for predicting oral absorption of a drug.
Toxicity risks (mutagenicity, tumorogenicity, irritation, reproduction) and
physicochemical properties (drug likeness and drug score) of compounds 9a–c were
calculated by the methodology developed by Osiris (Table 3).
The toxicity prediction using Osiris Property Explorer was shown in color
codes. Green color shows the low toxicity tendency, yellow shows the moderate
tendency, and red shows high tendency. The result of toxicity analysis of compounds
9a–c showed low toxicity tendency.
Drug likeness may be defined as a complex balance of various molecular proper-
ties and structure features that determine whether particular molecule is similar to the
known drugs. The drug-likeness value of standard and modified ligand shows the
fragment content of the drugs. If the drug-likeness values are increasing, then it has
the same fragment content with existing drugs.[45,46] Table 3 shows that the
drug-likeness values of 9a–c were lower than the standard drugs, with the exception
of amphotericin B. This result tells us that our thienopyridine phosphoramidates have
few fragments content of drugs.
The drug score values are the combination of solubility, molecular weight, log
P, drug likeness, and toxicity risk within one useful practical value. It could be used
for evaluating the potential of the drug candidate.[47] When the drug score is better,
then the compound has a better chance to be a drug candidate. The compounds 9a–c
showed better drug score than all standard drugs tested (Table 3).
Estimation of toxicity could be verified by using Toxtree v2.5.0, and the result
could be seen at Table 4. Toxtree v2.5.0 determines the toxicity level of compounds
based on Benigni and Bossa rules.[48] It stipulated that certain functional groups,
which have mutagenic or carcinogenic properties, could influence the toxicity.
Table 4 shows that compounds 9a–c have no tendency to be mutagenic and carcino-
genic, while benznidazole and nifurtimox have potential to be carcinogenic. Thus,
when Toxtree software result was compared with quantitative structure–activity
relationship (QSAR) analysis, the thieno[2,3-b]pyridine phosphoramidates presented
no mutagenic and carcinogenic potential.
In conclusion, the methodology for the nucleophilic aromatic substitution of the
chlorine atom in 4-substituted thieno[2,3-b]pyridine was successfully applied to ami-
noalkylphosphoramidates as the nucleophile. Greater yields were obtained when there
Table 3. Toxicity of thieno[2,3-b]pyridine phosphoramidates (9a–c) and standard drugs based on Osiris
Property Explorer
Reproductive
effective
Drug
likeness
Drug
score
Compound
Mutagenic
Tumorigenic
Irritant
9a
9b
Green
Green
Green
Green
Green
Red
Green
Green
Green
Green
Green
Green
Red
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Red
ꢂ22.47
ꢂ22.48
ꢂ23.70
ꢂ0.14
ꢂ54.74
ꢂ3.32
0.65
0.35
0.33
0.30
0.26
0.30
0.18
0.16
9c
Amphotericin B
Miltefosine
Benznidazole
Nifurtimox
Red
Red

3380
L. F. PEDROSA ET AL.
Table 4. Toxicity of thieno[2,3-b]pyridine phosphoramidates (9a–c) and standard drugs based on
Toxtree v2.5.0
Negative for
genotoxic
carcinogenity
Negative for
nongenotoxic
carcinogenity
Potential S. Typhiurium
TA 100 mutagen based
on QSAR
Potential
carcinogen
based on QSAR
Compound
9a
9b
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
9c
Amphotericin B
Miltefosine
Benznidazole
Nifurtimox
No
is a greater distance between the amino and phosphoramidates groups, suggesting that
the nucleophilic attack is somewhat dependent on the steric effect caused by the phos-
phoramidate group. This assumption is based on the crystal structures of compounds
9a and 9b in which hydrogen bonding contributed to approximate phosphoramidate
and the amino groups. In the case of compound 9a, the short alkyl chain between
phosphoramidate and thieno[2,3-b]pyridine groups leads to intramolecular and inter-
molecular hydrogen bonding P—O1—H—N2 atoms, increasing the steric hindrance.
The compounds 9a–c showed suitable druglike properties and are expected to
present good bioavailability profiles. The theoretical study of these molecules also
showed that they fulfilled the Lipinski rule of five and present drug scores better than
some standard drugs used as antiparasitics. The results encouraged us to invest in the
synthesis of new thienopyridine phosphoramidates and perform antiparasitic tests
in vitro and in vivo.
EXPERIMENTAL
Analytical-grade reagents and solvents were purchased from commercial
sources and used without further purification. Uncorrected melting points were
obtained with a Fisher-Johns apparatus. 1H, 13C, and 31P NMR spectra were recorded
on a Varian UP-300 spectrometer at 299.95, 75.42, and 121.42 MHz, respectively, with
tetramethylsilane (TMS) as internal standard or 85% H3PO4 as external standard. The
chemical shifts (d) are reported in parts per million (ppm) and the coupling constants
(J) are in hertz. Thin-layer chromatography (TLC) was carried out using silica-gel
F-254 glass plates (20 ꢄ 20 cm). Infrared spectra were recorded on a Perkin-Elmer
Spectrum One Fourier transform–infrared (FT-IR) spectrometer. High-resolution
mass spectra (EI-70 eV) were performed on a Varian MAT CH7 8500 direct inlet
instrument. The 4-chlorothieno[2,3-b]pyridine-5-carbonitrile (5)[6,15,31] and aminoalk-
ylphosphoramidates (6a–c) compounds[32,33] were prepared as previously reported.
General Procedure for Synthesis of the Thieno[2,3-b]pyridine
Phosphoramidates (9a–c)
4-Chlorothieno[2,3-b]pyridine-5-carbonitrile (5) (2.2 mmol) and the aminoalkyl-
phosphoramidate (6a–c) (4.4 mmol) were dissolved in THF (10 mL), and the reaction

THIENOPYRIDINE PHOSPHORAMIDATES
3381
mixture was heated at reflux until the disappearance of the starting 5 (9–12 h,
monitored by TLC). The mixture was poured onto ice, and the resulting solid (except
9c) was filtered off, washed with distilled water, and dried. Compounds 9a and b were
recrystallized from ethanol=water (1:3). Compound 9c was diluted with chloroform
and washed with water (3 ꢄ 10 mL). The solvent was evaporated under reduced
pressure, giving an oily product.
Diisopropyl 2-(5-cyanothieno[2,3-b]pyridin-4-ylamino)ethyl phosphora-
midate (9a). Yield 82%; pale brown solid; mp 130–131 ꢀC; IR (KBr, cmꢂ1): 3342
(m, nN-H), 3213 (m), 2982 (m), 2933 (m), 2208 (w, nCN), 1592 (s, dN-H),
=
1544(s), 1521 (m), 1450 (m), 1384 (w), 1358 (m), 1235 (m), 1194 (m, nP O), 1137
(m), 1099 (m), 1000 (s, nP-O), 842 (w), 775 (w), 707 (w); 1H NMR (CDCl3):
3
d ¼ 1.31 and 1.33 [2d, 12H, J(HH) ¼ 6.4], 2.08 (br s, 1H), 3.43 (m, 2H), 4.05 (m,
3
3
3
2H), 4.59 [dsep, 2H, J(HH) ¼ 6.3 and J(PH) ¼ 7.5], 7.46 [d, 1H, J(HH) ¼ 6.0],
8.04 [d, 1H, 3J(HH) ¼ 6.0], 8.45 (s, 1H), 8.86 (br s, 1H); 13C NMR (CDCl3):
d ¼ 23.66 and 23.59 [2d, 3J(PC) ¼ 5.3 and 4.7], 40.30 (s), 47.04 (s), 71.95 [d,
2J(PC) ¼ 5.9], 119.36 (s), 119.56 (s), 124.54 (s), 150.93 (s); 31P NMR (CDCl3):
d ¼ 9.84 (s); HRMS (EI): m=z [M þ 1] calcd. for C16H23N4O3PS: 382.12285. Found:
382.12280.
Diisopropyl 3-(5-cyanothieno[2,3-b]pyridin-4-ylamino)propyl phos-
phoramidate (9b). Yield 73%; pale brown solid; mp 125 ꢀC; IR (KBr, cmꢂ1):
3332 (m, nN-H), 3271 (m), 2978 (m), 2932 (m), 2210 (w, nCN), 1591 (s, dN-H),
=
1546 (s), 1437 (w), 1374 (w), 1220 (s), 1194 (m, nP O), 1130 (m), 1082 (w), 1018
1
(s), 988 (s, nP-O), 899 (w), 838 (w), 755 (w), 713 (m); H NMR (CDCl3): d ¼ 1.26
and 1.28 [2d, 12H, 3J(HH) ¼ 6.1], 1.85 [quin, 2H, 3J(HH) ¼ 5.4], 3.07 (m, 3H),
3
3
3
3.98 [dt, 2H, J(HH) ¼ 6.5], 4.55 [dsep, 2H, J(HH) ¼ 6.1 and J(PH) ¼ 7.3], 7.30
3
3
[d, 1H, J(HH) ¼ 6.0], 7.71 [d, 1H, J(HH) ¼ 6.0], 7.97 (br s, 1H), 8.33 (s, 1H);
13C NMR (CDCl3): d ¼ 23.73 and 23.79 [2d, J(PC) ¼ 4.8], 31.50 [d, J(PC) ¼ 3.6],
3
3
2
37.30 (s), 39.52 (s), 71.47 [d, J(PC) ¼ 6.0], 119.31 (s), 119.38 (s), 124.02 (s), 150.48
(s), 151.45 (s); 31P NMR (CDCl3): d ¼ 9.20 (s); HRMS (EI): m=z [M þ 1] calcd. for
C17H25N4O3PS: 396.13850. Found: 396.13850.
Diisopropyl 4-(5-cyanothieno[2,3-b]pyridin-4-ylamino)butyl phosphor-
amidate (9c). Yield 64%; yellow oil; IR (CH2Cl2, cmꢂ1): 3268 (m, nN-H), 3109
(m), 2977 (s), 2931 (s), 2208 (w, nCN), 1592 (m, dN-H), 1543 (s), 1437 (m), 1373
=
(m), 1225 (s), 1106 (m, nP O), 987 (s, nP-O), 896 (m), 836 (w), 775 (m), 736 (m),
1
3
702 (w), 665 (w); H NMR (CDCl3): d ¼ 1.26 and 1.28 [2d, 12H, J(HH) ¼ 6.3],
3
1.49 (br s, 1H), 1.65 (m, 2H), 1.82 [quin, 2H, J(HH) ¼ 6.5], 3.04 (m, 2H), 3.79
[dt, 2H, 3J(HH) ¼ 6.5 and 6.0], 4.55 [dsep, 2H, 3J(HH) ¼ 6.3 and 3J(PH) ¼ 7.5],
6.41 (br s, 1H), 7.30 [d, 1H, 3J(HH) ¼ 6.0], 7.58 [d, 1H, 3J(HH) ¼ 6.0], 8.38 (s,
1H); 13C NMR (CDCl3): d ¼ 23.63 and 23.70 [2d, J(PC) ¼ 4.6], 26.85 (s), 28.43 [d,
3
3J(PC) ¼ 5.8], 40.72 (s), 44.14 (s), 70.76 [d, 2J(PC) ¼ 5.2], 119.26 (s), 118.90 (s),
119.34 (s), 123.75 (s), 150.52 (s), 151.10 (s), 163.95 (s); 31P NMR (CDCl3):
d ¼ 7.79 (s), HRMS (EI): m=z [M þ 1] calcd. for C18H27N4O3PS: 410.15415. Found:
410.15420.

3382
L. F. PEDROSA ET AL.
X-Ray Structure Determination
The crystallographic data for compounds 9a and 9b were collected on an Enraf
Nonius Bruker Kappa CCD diffractometer, using graphite monochromatic MoKa
˚
radiation (k ¼ 0.71069 A). Final unit cell parameters were based on the fitting of
all reflections positions using DIRAX.[49] Collected reflections were integrated
using the EVALCCD program.[50] Empirical multiscan absorption corrections using
equivalent reflections were performed with the SADABS program.[51] The structure
solutions and full-matrix least-squares refinements based on F2 were performed with
the SHELXS-97 and SHELXL-97 program packages.[52] All atoms except hydrogen
were refined anisotropically. Hydrogen atoms were treated by a mixture of
independent and constrained refinement. Details of data collection and structure
refinement are summarized in Table S1. Selected distances and angles are given in
Tables 1, S2, and S3. CCDC ID 886689 (9a) and 886690 (9b) contains the supplemen-
tary crystallographic data for this article. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: þ44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
Computational Methods Applicable for Lipinski’s Rule and
Toxicity Prediction
The molecular properties of compounds were predicted in Molinspiration
software[53] using the SMILES format. These properties include molecular mass,
miLogP, TPSA, number of hydrogen-bond acceptors, number of hydrogen-bond
donors, nRotb, and number of Lipinski’s rule of five violations. The fragment-based
approach used by Molinspiration has proven to be reliable and is employed in
relevant scientific publications and chemical databases.
The OSIRIS Property Explorer[54] is an integral part of Actelion’s inhouse
substance registration system. It lets you draw chemical structures and calculates
on-the-fly various drug-relevant properties whenever a structure is valid. Prediction
results are valued and color coded. Green color shows the low toxicity tendency,
yellow shows the moderate tendency, and red shows high tendency.
Toxicology analysis was performed using software ToxTree v2.5.0[55] based on
the Benigni and Bossa rules for mutagenicity and carcinogenicity developed by
Romualdo Benigni and Cecilia Bossa from the Instituto Superiore in Sanita, Rome,
Italy, and approved by the European Chemical Bureau, Institute for Health and
Consumers Protection, European Commission Joint Research Centre (JRC) in 2008.
SUPPORTING INFORMATION
Crystal data and 1H, 13C, and 31P NMR spectra can be found via the
Supplementary Content section of this article’s Web page.
ACKNOWLEDGMENTS
`
We thank Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro
´
(FAPERJ), Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
´

THIENOPYRIDINE PHOSPHORAMIDATES
3383
´
(CNPq), and Coordenac¸a˜o de Aperfeic¸amento de Pessoal de Nıvel Superior (CAPES)
`
for financial support and the x-ray diffraction laboratory Laboratorio de Difrac¸a˜o de
Raio-X da Universidade Federal Fluminense (LDRX-UFF) for data collection.
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