Aqueous synthesis of N,S-dialkylthiophosphoramidates: Design, optimisation and application to library construction and antileishmanial testing

Article abstract of DOI:10.1039/c3ob27448a

We recently reported the use of PSCl₃ for the thiophosphorylation of alkylamines where the resulting N-thiophosphoramidate ions could be readily S-alkylated (Chem. Commun., 2011, 47, 6156-6158.). Herein we report the development of this methodology using amino acid, amino sugar, aminonucleoside and aniline substrates. The hydrolysis properties of N-thiophosphoramidate ions and their reactivities towards alkylating agents are also explored. In addition, we demonstrate the application of our approach to the preparation of a small library of compounds, including quinoline-based N,S-dialkylthiophosphoramidates which were tested for antileishmanial activity.

Full text of DOI:10.1039/c3ob27448a

Organic &
Biomolecular Chemistry
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Aqueous synthesis of N,S-dialkylthiophosphor-
amidates: design, optimisation and application to
Cite this: Org. Biomol. Chem., 2013, 11,
2660
library construction and antileishmanial testing†
Milena Trmčić,a Frances L. Chadbourne,a Paul M. Brear,a Paul W. Denny,b,c
Steven L. Cobba and David R. W. Hodgson*a
We recently reported the use of PSCl3 for the thiophosphorylation of alkylamines where the resulting
N-thiophosphoramidate ions could be readily S-alkylated (Chem. Commun., 2011, 47, 6156–6158.).
Herein we report the development of this methodology using amino acid, amino sugar, aminonucleoside
and aniline substrates. The hydrolysis properties of N-thiophosphoramidate ions and their reactivities
towards alkylating agents are also explored. In addition, we demonstrate the application of our approach
to the preparation of a small library of compounds, including quinoline-based N,S-dialkylthiophosphor-
amidates which were tested for antileishmanial activity.
Received 18th December 2012,
Accepted 28th February 2013
DOI: 10.1039/c3ob27448a
www.rsc.org/obc
Recently, we reported a simple aqueous method for the
Introduction
preparation of N,S-thiophosphoramidates.11 These phospho-
Phosphate esters are key intermediates in the transmission of diester mimics, with their N- and S-bridges, were assembled
genetic material and many other critical cellular processes. through the electrophilic action of the reactive phosphorylat-
Structural analogues of these systems have been used widely ing agent PSCl3 on nucleophilic primary alkylamines followed
as tools for determining enzyme mechanisms and as inhibi- by S-alkylation of the resulting N-thiophosphoramidate ions—
tors or activators of these enzymes. Sulfur-based analogues an approach that builds on our established use of reactive P
have been used in place of phosphodiesters to both increase species in aqueous systems.12–14 Here we describe the develop-
and decrease the rate of ester bond cleavage. Accelerated clea- ment of our strategy for aminothiophosphorylation, including
vage rates are offered by S-bridging systems,1 where the thio- the use of alkyl, aryl, amino acid, amino–sugar and amino-
late leaving group departs more readily than its alkoxide nucleoside substrates. We describe kinetic studies on the
analogue, whereas reduced cleavage rates are seen for non- pH-dependent hydrolysis properties of N-thiophosphoramidate
S-bridging systems.2 Nitrogen-bridging phosphodiester mimics, ions which were used to inform subsequent optimisation of
where N-protonation becomes possible, have also been S-alkylation steps. Studies on several S-reactive electrophile
generated and studied.3–10 A combination of N- and S-bridging species and alkylation conditions are also reported, along with
systems have seen application in the form of phosphate tri- our efforts towards using bromoacetamides as generic amine-
ester mimics that show antiviral activity. These uncharged, derived alkylating agents. Finally, our thiophosphorylation–
nucleoside-based thiophosphoramidates serve as prodrugs alkylation conditions were applied to a library of lipophilic
which traverse cell membranes, however, within the cell, pro- amines which were then alkylated with a quinoline derivative
grammed hydrolysis occurs to reveal nucleoside monophos- before being screened for activity against Leishmania mexicana,
phates that go on to interfere with viral replication.
a
causative agent of the Neglected Tropical Disease
leishmaniasis.15
aDepartment of Chemistry, Science Laboratories, Durham University, South Road,
Durham, DH1 3LE, United Kingdom. E-mail: d.r.w.hodgson@durham.ac.uk
bSchool of Medicine and Health, Queen’s Campus, Stockton-on-Tees, TS17 6BH,
United Kingdom
Combined thiophosphorylation-S-alkylation
of alkyl-, aryl- and biomolecule-derived
amines
cSchool of Biological and Biomedical Sciences, Science Laboratories, Durham
University, South Road, Durham, DH1 3LE, United Kingdom
We demonstrated that simple alkylamines are effective sub-
strates for PSCl3 under aqueous conditions in the presence of
NaOH. The resulting N-thiophosphoramidate ions can then be
†Electronic supplementary information (ESI) available: Details of kinetic
studies; 31P NMR, 1H, 31P and 13C NMR spectra of reported compounds;
summary of biological testing data. See DOI: 10.1039/c3ob27448a
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Scheme 1 Aqueous N-thiophosphorylation and S-alkylation.
Fig. 1 Structural resemblance of amide hydrolysis and amino acid-N-
thiophosphoramidates.
Scheme 2 N-Thiophosphorylation and S-alkylation of aniline.
Scheme 3 N-Thiophosphorylation–S-alkylation of phenylalanine.
alkylated effectively using a range of soft alkylating agents
(Scheme 1).
With the aim of broadening the scope of amine substrate
used in this method we explored the thiophosphorylation and
S-alkylation of aniline, unprotected phenylalanine, glucos-
amine and two 5′-amino-5′-deoxynucleosides.
structure that has been shown to be an effective N-phosphory-
lating agent for amino acids. In addition to N-phosphorylation,
in the context of an amino acid, the carboxylate group acts as
an internal nucleophile, displacing pyrophosphate ion and
forming a cyclic mixed anhydride-phosphoramidate species.
Cyclic phosphate esters show enhanced electrophilicity over
their acyclic counterparts, and in this guise, the cyclic mixed
anhydride-phosphoramidates show electrophilicity towards
water at the phosphoryl centre and amines at the carbonyl.
Histidine side chains of proteins have also been successfully
modified with thiophosphorylating agents (PSCl3 and thiophos-
phoramidate ion) in order to prepare more hydrolytically
stable analogues of phosphohistidyl proteins that are inter-
mediates in a variety of signalling enzymes.16–18
With these ideas in mind, we hoped to apply PSCl3 towards
the primary amino function of phenylalanine and alkylate the
resulting thiophosphoramidate ion to produce carboxamide
hydrolysis transition state analogues (Fig. 1).
Using 1.0 eq. phenylalanine, 7.0 eq. NaOH and 1.4 eq.
PSCl3, 85% N-thiophosphorylation was observed by 31P NMR
spectroscopy (Scheme 3) after removal of inorganic thiopho-
sphate through selective precipitation. The remainder of the
P-containing impurities included N-phosphoramidate (∼10%)
and several unidentified species.
S-Alkylation was attempted in D2O using methyl iodide,
however, only 24% conversion to the N,S-dialkylthiophosphor-
amidate 4 was observed. The remainder of the P-containing
materials included significant quantities of the N-phosphor-
amidate and phosphate ion.
Mass spectrometric analysis of the N-thiophosphorylation
mixture also revealed significant quantities of N-phosphor-
amidate 5 plus Phe–Phe dimer 6, however, the analysis was
performed under acidic conditions, which were likely to
encourage desulfurisation.
Aniline
We employed a 1.2 : 1 ratio of aniline 1 to PSCl3 followed by
alkylation of the expected N-thiophosphoramidate ion using
bromoethanol (Scheme 2). Thiophosphorylation proceeded to
a reasonable extent of 62% as determined by 31P NMR spectro-
scopy, with the key signal for thiophosphorylated amines
usually appearing in the shift range 40–45 ppm. After addition
1
of bromoethanol, a combination of H and 31P NMR methods
revealed ∼47% conversion to the N,S-thiophosphoramidate 2
(δ ∼ 20–25 ppm), and 32% of S-alkylated thiophosphate ion 3
(δ ∼ 15–20 ppm). The remainder of the product mixture was
predominantly aniline 1 (19%). The use of higher con-
centrations of aniline may have improved conversion to the
N-thiophosphoramidate, and our studies with morpholine
have shown that despite the increased potential for bis- and
tris-aminolysis of PSCl3, this is likely possible. However, on the
basis of this preliminary result, we did not pursue this
optimisation.
Phenylalanine
Post-translational phosphorylation of proteins represents a
major signalling pathway, and access to phosphoproteins and
their analogues supports the delineation of these key pro-
cesses. In addition, phosphorylation of the carboxyl group of
amino acids serves to activate the carbonyl group for substi-
tution during coded protein biosynthesis. Furthermore, phos-
phonamide systems have been widely exploited as transition
state mimics for the attack of water upon the carbonyl of
amides (Fig. 1). To date, a limited number of examples of
aqueous amino acid phosphorylations have been reported.
Metatriphosphate ion possesses an activated anhydride
Taken together, these pieces of evidence strongly support
the idea of intramolecular assistance of the carboxyl group in
the decomposition of the S-alkylated thiophosphoramidate
(or the protonated thiophosphoramidate ion), where the cyclic
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Scheme 4 Possible intramolecular reactions of phenylalanine-based thiophos-
phoramidate systems.
mixed anhydride intermediate 7 likely facilitates the formation
of several of the decomposition products (Scheme 4).
Scheme 5 N-Thiophosphorylation–alkylation of glucosamine and potential
pathways for decomposition.
Glucosamine
Phospho-sugar systems play many biological roles and we
hoped that we would be able to gain access to phospho-sugar
mimics using our approach. Glucosamine was chosen as a
readily available model substrate for our preliminary study.
Others have investigated aqueous sugar phosphorylation pro-
cedures using metatriphosphate ion and its imino analogue,
however, yields were low.28,29 The preparation of sugar phos-
phoramidates that go on to afford a phosphosugars has also
been investigated, and good yields (79%) were reported.30
A preliminary thiophosphorylation experiment using a 1 : 1
ratio of glucosamine 8 to PSCl3 gave a rewarding ∼90% con-
version (estimated from a signal ∼45.5 ppm in the 31P NMR
spectrum) to the N-thiophosphoramidate 9. Over a time course
of ∼1 h, however, this signal diminished, with new signals
appearing at similar chemical shifts. At present, we are not
able to assign these, however, they are consistent with thiopho-
sphoryl groups that have not been S-alkylated. Indeed, they
may represent phosphorothioates that have arisen through
dialkylthiophosphoramidates, which appear to be stable under
the reaction conditions. A tentative decomposition mechanism
for the glucosamine system is presented in Scheme 5. The key
difference between this system and the Phe system is the
potential for intramolecular acid catalysis via the 1-OH group,
which, as an acetal, could act as an acid at the relatively high
pHs used for thiophosphorylation (and alkylation). Smaller
signals ∼10–12 ppm are consistent with N-phosphoramidates
that result from desulfurisation processes, where the two
signals may signify the α- and β-anomers. On the basis of
these preliminary results, we did not explore this system
further, however, delivery of such a reactive phosphoryl donor
to an enzyme active site may offer a useful tool for enzyme lab-
elling or capture, and may also offer uses as a synthetic phos-
phorylation tool.
5′-Amino-5′-deoxyguanosine and 5′-amino-5′-deoxyadenosine
intramolecular isomerisation. Despite this process, we pro- Nucleoside phosphates are ubiquitous in biological systems,
ceeded with S-alkylation using MeI. This led to a majority of and a range of N-containing and S-containing phosphate
the P-containing product mixture being converted to S-methyl- mimics have been reported, with uses in mechanistic studies
ated inorganic phosphate ion 10. The large proportion of N–P and antisense/siRNA applications. We have already reported
bond scission suggests that once alkylated, intramole- the alkylation of N-thiophosphoramidate using a nucleoside-
cular reaction facilitates this cleavage, unlike simple N,S- 5′-iodide, and reasonable conversions were observed.
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Scheme 6 N-Thiophosphorylation–alkylation on 5’-aminonucleosides.
Alkylation with the nucleoside-5′-iodide, however, proved to be
very slow in comparison to other alkylating agents (see below).
With this in mind, we sought to explore the N-thiophosphory-
lation of 5′-amino-5′-deoxynucleoside substrates and their sub-
sequent alkylation.
We prepared adenosine and guanosine aminonucleosides
11a–b using established procedures. The adenosine system
11b was isolated as its hydrochloride salt, thus an additional
equivalent of NaOH was employed during thiophosphoryla-
tion. Alkylations were then performed using MeI, and, in both
cases, the S-alkylated aminonucleoside-N-thiophosphor-
amidates 12a–b were formed at conversions levels ∼70%. Given
that unprotected nucleosides were employed, this level is
impressive, however, chromatographic purification was necess-
ary (see ESI†) in order to confirm the identity of all the reac-
tion products (Scheme 6).
Fig. 2 31P NMR spectroscopy study of the hydrolysis of ethanolamine-N-thio-
phosphoramidate ion as a function of pH. Red circles represent rate constants
for the disappearance of ethanolamine-N-thiophosphoramidate ion (closed,
stronger buffers; open, weaker buffers); black triangles represent the appear-
ance of phosphate ion (a similar trace for the disappearance of thiophosphate
ion was also observed; not shown); and blue squares represent the appearance
of ethanolamine-N-phosphoramidate ion.
The desired thiophosphoramidate products eluted
∼35–40 min which corresponded to ∼100–140 mM TEAB. The
areas under the absorbance–elution time profiles were also
used to estimate conversion to the desired products, and the
values were in agreement with the observations from 1H and
31P NMR spectroscopies.
Scheme 7 Hydrolytic
breakdown
products
of
ethanolamine-N-
thiophosphoramidate.
S-Alkylation
In order to achieve effective alkylation, we explored the effects
of pH, stoichiometry, reaction time and temperature on model
substrates. In addition, we explored the kinetics of alkylation
of a range of thiophosphoramidate ions and alkylating agents.
The stability of the S-alkylated thiophosphoramidates was also
explored. These results are summarised in the following sub-
sections.
to enable a deuterium lock signal to be used. The use of 4 M
buffer solutions, ensured the pH changes observed during
kinetic experiments were small, however, the presence of large
concentrations of sodium ions caused problems with the
measurement of pH. This manifested itself in the form of devi-
ation from the expected gradient of −1 in the log kobs-pH plot
for pH > 8. On this basis, some of the experiments at higher
pHs were repeated using 0.5 M buffers. Under these con-
ditions, greater changes in pH (0.2–0.5) were observed during
the courses of the kinetic experiments, however, a gradient of
−1 was observed for the log kobs-pH data at higher pHs (Fig. 2).
Caution should also be taken in terms of the interpretation of
measured pH values where the extent of deuteriation within
the buffer is not clearly defined.
Stability of N-thiophosphoramidate ions
In line with N-phosphoramidates, we expected N-thiophos-
phoramidates to display greater stability at higher pHs. In
order to explore this idea, we conducted 31P NMR kinetic
studies using ethanolamine-N-thiophosphoramidate 13 as sub-
strate. The use of 31P NMR spectroscopy allowed us to monitor
the decomposition of the ethanolamine-N-thiophosphor-
amidate and to gain insight into the identities of the resulting
hydrolysis products through the use of chemical shift and
signal multiplicity data. The substrate was dissolved in 4 M or
0.5 M buffer, the water was removed by lyophilisation and the
residue was redissolved in D2O. This provided solutions where
∼90% of the labile protons had been exchanged for deuterium
Closer analysis of the 31P NMR spectra shows that in
addition of P–N scission to give amine and inorganic thiopho-
sphate ion, desulfurisation of both the N-thiophosphor-
amidate substrate 13 and thiophosphate ion occurs (Scheme 7)
to give N-phosphoramidate 14 and phosphate ion, respecti-
vely.9,10,19 Desulfurisation of the N-thiophosphoramidate
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ion, however, was only detectable for 7 < pH < 9, whereas
desulfurisation of thiophosphate ion was seen across the
profile. Rate constants for the processes discussed above have
been estimated, however, the compromises made in terms of
the use of buffers to facilitate the use of 31P NMR spectroscopy,
mean that these rate constants should only be considered on
an order-of-magnitude basis. On the pH plateaux, disappear-
ance of thiophosphoramidate shows a rate constant of ∼4 ×
10−2 s−1; appearance of phosphate, 5 × 10−4 s−1; and appear-
ance of phosphoramidate, ∼6 × 10−5 s−1. Taken together,
however, these data give clear evidence that N-thiophosphor-
amidate species display similar pH-reactivity properties to
their oxy-analogues, and the use of higher pH would appear to
be the most reliable pathway towards S-alkylation. In addition,
these data align well with the findings of Ora et al. and their
studies on closely related systems.
Scheme 8 (A) Disconnection strategy for thiophosphoramidate-bromoacetate
ester ligation of two amines. (B) Structural resemblance of thiophosphoryl-acet-
amide system to pyrophosphate.
Stability of N,S-dialkylthiophosphoramidates
To gain an appreciation of the stability of N,S-dialkylthiopho-
sphoramidates, we performed 31P NMR spectroscopy-based
studies on N-benzylamino-S-n-propylthiophosphoramidate at
pH ∼ 7.5 and ∼5.2. The pH of 7.5 was chosen to be close to
physiological pH, whereas the pH 5.2 provides a situation
where N-protonation is more likely, and reactivity is expected
to be higher. In addition, the lower pH aligns with the con-
ditions used for amastigote testing, which will be discussed
below. The samples were incubated at 37 °C and 31P NMR
spectra were recorded periodically. No changes in the forms of
the spectra were observed over the course of 16 h. On the basis
of these results, where we would expect to be able to detect 5%
degradation reliably using the NMR method, we predict half-
lives >200 h in both cases. Whilst this picture suffices for the
development of our synthetic procedures, further detailed
kinetic studies will be required.
Scheme 9 Amine-amine ligation via thiophosphorylation-bromoacetate ester
cross-linking.
amine, RNH2, was either the model system, allylamine, or the
more challenging 5′-amino-5′-deoxyguanosine (Scheme 9).
After each reaction, excess amine RNH2 was removed by
increasing pH followed by extraction with organic solvent. The
pH was then reduced to facilitate protonation of the phenolate
leaving groups by extraction into organic solvent. In all cases,
the majority of material was converted to the desired thio-
phosphoramidate-acetamide products 16 and 17.
In order to confirm the identity of the guanosine-derived
product 16, ion exchange chromatography was carried out. As
seen for the N-thiophosphorylated aminonucleoside systems
(see above), the desired thiophosphoramidate product 16
eluted ∼35–40 min which corresponded ∼90 mM TEAB
(see ESI†). The conversion level estimated by measuring
the area under the absorbance curve in the elution profile
correlated well with observations from 1H and 31P NMR
spectroscopies.
Bromoacetamides as alkylating agents
In order to expand the range of potential alkylating agents
available for elaboration of N-thiophosphoramidate ions, we
explored the use of a heterobifunctional cross-linking agents.
We envisioned 15 being able to react selectively with thiophos-
phoramidate ions to produce activated acylating agents 16 that
could be further reacted with readily accessible amines to
produce mixed phosphoryl–acyl systems 17 that may serve as
pyrophosphate mimics (Scheme 8). Our earlier experiences
with a thiophosphate anion-based system (uridine-5′-mono-
phosphorothioate, UMPS), suggested that this strategy could
offer a convenient aqueous route to these species.20
Based on our earlier work with thiophosphates, we per-
formed exploratory studies on the use of p-nitrophenyl-,
m-nitrophenylandphenylbromoacetateesters15a–crespectively.
Our aim was to balance hydrolysis of the activated ester
against the desired aminolysis process by tuning the reactivity
of the phenolate leaving group. We used benzylamine as a
model substrate for thiophosphorylation given that we had
observed this process to proceed quantitatively. The second
These preliminary studies illustrate that the thiophosphory-
lation-bromoacetate route could offer a simple route towards
nucleoside-based systems. Further optimisation of conditions,
reaction times and the choice of phenolate leaving group
should facilitate improvements.
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systems served to illustrate the usage of a reactive benzyl
system and a simple alkyl system. The quinolines, on the other
hand were designed by analogy with quinoline-based sulf-
amidates that have been successfully applied as anti-parasite
agents. The syntheses of the sulfonamides were, however, by
way of organic solvent-based procedures where laborious puri-
fication procedures were required. We hoped that the similar
geometric properties of the thiophosphoramidate group may
offer an alternative to the sulfonamide where product mixture
could be used directly from aqueous synthetic procedures
without isolation.
Scheme 10 Thiophosphoryl systems and alkylating agents used in S-alkylation
kinetic study.
Nucleophilicity of thiophosphoryl systems
During our alkylation studies, we observed that some
alkylations appeared more sluggish than others, thus we
sought to explore these observations through kinetic studies.
In addition to being sensitive to the nature of the electrophile,
we expected the kinetics of alkylation to vary as a function of
the nature of the N-alkyl portion of the thiophosphramidate.
We studied the progress of a series of alkylation reactions
using 31P NMR spectroscopy using ethanolamine-N-thiophos-
phoramidate 13, benzylamine-N-thiophosphoramidate 18,
and, as a comparison, inorganic thiophosphate ion as nucleo-
philes. The added electrophiles were bromoethanol and
5′-deoxy-5′-iodoguanosine 1 (Scheme 10).
Testing antileishmanial activities
Quinoline-substituted sulfonamides have been reported as
potential antileishmanial agents (Fig. 3).22,23
Owing to the close structural homology of the thiopho-
sphoramidate and sulfonamide groups, we prepared
quinoline-based thiophosphoramidate derivatives 20–33a. In
addition, phosphorothiolate-quinoline system 34 was prepared
as a control that represents the common hydrolysis product
expected from P–N scission. We tested these systems for
activity against both mammalian stage amastigote and insect
stage promastigote forms of the Trypanosomatid Leishmania
mexicana using established protocols.24 Unfortunately, there
were no clear signs of activity of these agents. In the case of
amastigotes, we found that the quinoline systems were quite
sensitive to the acidic nature of the specialist growth media,
and showed significant decomposition over a timescale of
hours. This contrasts with our findings for other systems, and
we attribute this difference to the possibility of intramolecular
nucleophilic catalysis in the quinolines (Scheme 11).
Alkylations were performed in the presence of a significant
excess of alkylating agent to facilitate first order kinetic
analyses. Using the reactive bromoethanol system, alkylations
proceeded rapidly, thus we were unable to derive kinetic
data. With the less reactive nucleoside system, however, bi-
molecular rate constants, k2, of 4 × 10−5, 2.5 × 10−4 and 3.3 ×
10−4
M
−1 s−1 were obtained for ethanolamine-N-thiophosphor-
amidate 13, benzylamine-N-thiophosphoramidate 18 and in-
organic thiophosphate ion, respectively. These data confirm
that the nature of the substituent on the thiophosphoryl group
can have a significant effect on alkylation kinetics.
The nucleophilicity of thiolate ions can be measured quan-
titatively, and we would expect these values to be similar
in nature to thiocarboxylate systems studies by Mayr and co-
workers.21 We are currently exploring these values.
Promastigote testing also failed to demonstrate antileish-
manial activity, thus we must conclude that N-alkyl-S-(methyl-
ene(8-quinolyl)) thiophosphoramidates are not effective
against Leishmania mexicana, despite their structural resem-
blance to successful sulfonamide compounds.
Conclusions
Aqueous aminothiophosphorylation offers clean conversion to
thiophosphoramidate anions when used in conjunction with
nucleophilic, simple alkylamines, however, aryl systems have
proven less successful. Unprotected amino acid, sugar and
nucleoside systems showed varying degrees of effectiveness,
Preparation of N,S-dialkylthiophosphor-
amidate libraries using lipophilic
alkylamines
To prove the general applicability of the method, we prepared with the aminonucleoside systems showing significant
a small generic library of N,S-dialkylthiophosphoramidates promise and scope for improved performance. In these cases,
20–33a–c in a simple, rapid manner where the only form of thiophosphorylations proceeded, in most cases, smoothly,
purification was extraction of excess alkylating agent followed however, on S-alkylation, decomposition was seen. In all cases,
by removal of the aqueous solvent. All amines were hydro- mechanisms involving intramolecular assistance can be postu-
phobic in nature, and some of the reaction mixtures were lated, and it is these that we believe lead to the decomposition
heterogeneous (Table 1).
in these systems.
Alkylating agents were represented by benzyl chloride,
The straightforward assembly of simple lipophilic systems
n-propyl iodide and a quinoline system. The simple alkyl using PSCl3 allowed us to rapidly assemble a library of
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Table 1 Preparation of
a
library of N,S-dialkylthiophosphoramidates and
Table 1 (Contd.)
control compound
Entry
Product
Conversion level (%)
Entry
Product
Conversion level (%)
29
a 94a, 96b
b 95a, 92b
20
a 81a, 60b, 54c
b 97a, 97b, 92c
c 98a, 100b, 100c
30
31
a 87a, 75b
c 95a, 82b
21
22
a 79a, 80b
b 98a, 96b
c 98a, 95b
a 75a, 78b
c 79a, 87b
a 75a, 68b
b 95a, 97b
c 98a, 98b
32
33
a 81a, 67b
c 91a, 90b
23
a 90a, 90b
b 92a, 94b
c 93a, 94b
a 90a, 92b, 84c
c 96a, 96b, 100c
95a, 93b
24
25
a 93a, 90b
b 94a, 97b
c 97a, 98b
34
a 61a, 80b
b 99a, 100b
c 97a, 94b
a Determined by 31P NMR spectroscopy. b Determined by 1H NMR
spectroscopy. c Determined by 19F NMR spectroscopy.
compounds, and, although the quinoline systems presented
specific stability issues, the approach proved effective in facili-
tating swift access to aqueous solutions of library molecules
that were amenable to biological testing without needing
extensive purification.
26
a 65a, 65b
b 91a, 90b
c 67a, 60b
27
28
a 91a, 95b
a 93a, 96b
Experimental
Attempted thiophosphorylation-alkylation of aniline
(towards 2,3)
Aniline (1.2 eq., 251 μL, 2.76 mmol) was mixed with aqueous
sodium hydroxide (5 eq. of a 1 M aqueous solution, 11.5 mL,
11.5 mmol) and water (1.48 mL) in a 50 mL round bottomed
flask with indentations aimed towards inducing turbulent
mixing. The mixture was cooled on an ice-water bath,
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thiophosphoramidate (0.5 mmol, 130.5 mg) was dissolved in
D2O (0.5 mL) and MeI (0.5 mmol, 31 μL) was added directly to
the NMR. The sample was subjected to NMR analysis after
20 h. Analysis after thiophosphorylation; δH(400 MHz; D2O)
7.34–7.02 (5 H, m, Ar-H), 3.76 (1 H, ddd, J 12.4, 7.9, 4.5, CH),
3.06 (1 H, dd, J 13.1 and 4.5, CHH), 2.79 (1 H, dd, J 13.1 and
Fig. 3 Quinoline-based sulfonamides used in antileishmanial testing studies.
3
7.9, CHH); δP(162 MHz; D2O) 42.3 (d, JH–P 12.5, NPS); m/z
(ES+) 262.03 (M + H+); m/z (ES−) 244.0383 (phosphoramidate
i.e. loss of S, (M − H). C9H11NO5P requires 244.0380). Analysis
after addition of MeI; δH(400 MHz; D2O) 7.53–7.05 (6 H, m,
Ar-H), 3.90–3.68 (1 H, m, CH), 3.21–3.07 (2 H, m, CH2), 1.67
3
(3 H, d, J 13.3, SCH3); δP(162 MHz; D2O) 25.1 (d, JH–P 12.8,
NPS).
Attempted thiophosphorylation-alkylation of glucosamine
(towards 9, 10)
Scheme 11 Potential intramolecular mechanism accounting for the instability
of quinoline-based thiophosphoramidates.
Glucosamine hydrochloride (1.0 eq., 496 mg, 2.3 mmol) was
dissolved in aqueous sodium hydroxide (6 eq. of a 1 M
aqueous solution, 13.8 mL, 13.8 mmol) in a 50 mL round
bottomed flask with indents in an ice bath. Thiophosphoryl
chloride (1.0 eq., 232 μL, 2.3 mmol) in THF (7 mL) was added
dropwise to the mixture over the course of 10 min. After 1 h of
stirring, the conversion to thiophosphoramidate was estimated
by 31P NMR spectroscopy MeI (2.0 eq., 4.6, 286 μL) was added
and the mixture was stirred for a further 1 h and subjected to
31P NMR spectroscopy analysis to assess S-alkylation. Analysis
after thiophosphorylation; δP(162 MHz; D2O) 45.6 (NPS).
After addition of MeI, the majority of material appeared to be
converted to S-methylthiophosphate 10; δP(162 MHz; D2O)
19.3 (d, 3JH–P 11.3, OPSMe).
thiophosphoryl chloride (1.0 eq., 232 μL, 2.3 mmol) in THF
(7 mL) was added dropwise over the course of 10 min. and the
mixture was stirred for an additional 15 min. Bromoethanol
(2 eq., 326 μL, 4.6 mmol) was added and the mixture was
stirred for 22 h while maintaining pH ∼ 9 through periodic
additions of 1 mL aliquots of 1 M NaOH solution. The mixture
was then extracted with diethyl ether (3 × 10 mL) to remove
excess aniline, PSCl3, THF and bromoethanol, and the
aqueous layer was concentrated by lyophilisation before being
subjected to analysis. The conversion to N-thiophosphor-
amidate was estimated by 31P NMR spectroscopy before
addition of bromoethanol (see ESI†). After addition of the
alkylating agent, conversion was estimated using 31P NMR
spectroscopy (47%) and 1H NMR spectroscopy (41%). The
other impurities present were aniline 19% and alkylated in-
organic thiophosphosphate 31% by both 31P NMR and 1H NMR
spectroscopy. δH(400 MHz; D2O) 7.26 (2 H, t, J 7.9, m-Ar-H),
7.09 (2 H, d, J 8.0, o-Ar-H), 6.99–6.90 (1 H, m, p-Ar-H), 3.56
(2 H, t, J 6.4, CH2OH), 2.78–2.65 (2 H, m, SCH2); δP(162 MHz;
Thiophosphorylation of 5′-amino-5′-deoxyguanosine or
5′-amino-5′-deoxyadenosine and alkylation with MeI or BnCl
5′-Amino-5′-deoxyguanosine25,26 (1 eq., 0.23 mmol) or
5′-amino-5′-deoxyadenosine dihydrochloride27 (1 eq., 0.23 mmol)
was dissolved in a mixture of aqueous sodium hydroxide (5 eq.
of a 1 M solution, 1.15 mmol for G; 7 eq., 1.61 mmol for A)
and water (148 μL for G, 0 μL for A) in a 50 mL round
bottomed flask with indents in an ice bath. Thiophosphoryl
chloride (1 eq., 23.2 μL, 0.23 mmol) in THF (0.7 mL) was
added dropwise to the aqueous solution over the course of
10 min. and the mixture was then stirred for a further 1 h.
Methyl iodide (2 eq., 28.6 μL, 0.46 mmol) and additional
aqueous sodium hydroxide solution (1 eq.) were added to the
flask and stirring was continued for 1 h. The excess of alkylat-
3
D2O) 18.7 (t, JH–P 13.4, NPS); δC(101 MHz; D2O) 141.7, 129.6,
3
3
121.3, 118.2 (d, JC–P 6.7, CHCNH), 61.6 (d, JC–P 4.7, CH2OH),
32.5 (SCH2); m/z (ES−) 232.0203 (M − H. C8H11NO3PS requires
232.0203).
Attempted thiophosphorylation-alkylation of phenylalanine
(towards 4)
D/L-Phenylalanine (1 eq., 2.3 mmol, 380 mg) was dissolved in ing agent was removed by ether extraction (3 × 10 mL). The
aqueous sodium hydroxide (7 eq. of a 5 M aqueous solution, residual aqueous solution was then lyophilised and the resi-
3.22 mL, 16.1 mmol) in a 50 mL round bottomed flask with dues were analysed (see crude 1H and 31P NMR spectra in
indents in an ice bath. Thiophosphoryl chloride (1.4 eq., ESI†). The crude samples were dissolved in a 50 mM TEAB
327 μL, 3.22 mmol) in THF (4 mL) was added dropwise to the buffer, pH 7.5 (5 mL) and purified on DEAE Sepharose® FF
mixture over the course of 10 min. After 1 h of stirring, in- column (50 mL, 10 × 3 mm, 3 mL min−1), running TEAB
organic thiophosphate ion arising from hydrolysis of the buffer gradient 50–200 mM. Fractions were pooled and
excess PSCl3 was removed by applying methanol precipi- lyophilised before confirmation of their identities by 1H and
tation.25 The residual supernatant solution was concentrated 31P NMR spectroscopies. The triethylammonium salts of the
in vacuo before being freeze-dried to remove water and compounds were dissolved in water (5 mL) and passed
being subjected to NMR analysis. The crude phenylalanine through a Na-Dowex® 50 W × 2, 200–400 (50 mL, 30 × 2 mm,
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3 mL min−1) column, with water as the mobile phase. The frac- the spectra, set to have the value 1, were plotted as a pseudo
tions containing products, detected via UV trace (254 nm), first order function of time and least squares fittings were per-
were collected, lyophilised and spectroscopic analyses were formed against an exponential decay curve It = I0 e−kt
.
performed on the residues.
Bromoacetamide cross-linker
11a.
Use of benzylamine-N-thiophosphoramidate 18 with phenyl-
bromoacetates 15a–b and allyl amine or 5′-amino-5′-deoxy-
guanosine. Benzylamine (1 eq., 25 μL, 0.23 mmol) was
thiophosphorylated using our established procedure. Allyl-
amine (2 eq., 32 μL, 0.46 mmol) or 5′-amino-5′-deoxyguanosine
(1 eq., 65 mg, 0.23 mmol) was added to aqueous/THF solution
of the thiophosphorylated benzylamine and mixed for several
minutes, before the phenylbromoacetate ester (1 eq.,
0.23 mmol) was added. After 15 minutes of vigorous stirring,
the pH of the mixture was adjusted using 50 mM hydrochloric
acid to the approximately the pKa of the phenol leaving group.
The solution was extracted with ethyl acetate (3 × 10 mL), the
pH was adjusted to pH 9 and the extraction was performed
using chloroform (3 × 10 mL) in an attempt to remove excess
amine. The aqueous sample was lyophilized and the dry solid
was analysed and purified (nucleoside).
δH(700 MHz; D2O) 7.71 (1 H, s, 8-H), 5.66 (1 H, d, J 7.8, 1′-CH),
4.98–4.94 (1 H, m, 2′-CHOH), 4.31–4.28 (1 H, m, 3′-CHOH),
4.22–4.19 (1 H, m, 4′-CH), 3.06–2.99 (2 H, m, 5′-CH2), 1.97 (3 H,
d, J 13.0, CH3S); δP[1H](283 MHz; D2O) 26.4–26.1 (m, NHPS);
δC(176 MHz; D2O) not assigned owing to low spectrum inten-
sity; m/z (ES−) 391.0594 (M − H. C11H16N6O6PS requires
391.0595).
11b.
17.
δH(700 MHz; D2O) 8.20 (1 H, s, 2-H), 8.08 (1 H, s, 8-H), 5.88
(1 H, d, J 6.6, 1′-CH), 4.76 (1 H, t, J 5.4, 2′-CHOH), 4.33–4.29
(1 H, m, 3′-CHOH), 4.17–4.13 (1 H, m, 4′-CH), 3.13–3.00 (2 H,
m, 5′- NH2CH2), 1.95 (3 H, d, J 13.1, CH3S); δP[1H](283 MHz;
D2O) 26.1–25.8 (m, NHPS); δC(176 MHz; D2O) 155.6, 152.8,
δH(400 MHz; D2O) 7.45–7.22 (5 H, m, C6H5), 5.85–5.74 (1 H, m,
CH2vCH), 5.20–5.10 (2 H, m, CH2vCH), 4.00 (2 H, d, J 10.9,
CH2NH), 3.72 (2 H, dt, J 5.1 and 1.6, NHCH2), 3.33 (2 H, d, J
12.9, SCH2); δP[1H](162 MHz; D2O) 22.0–21.6 (m, NHPS);
δC(101 MHz; D2O) 172.4 (d, 3JC–P 3.6, CvO), 140.6 (d, 3JC–P 7.6,
CCH2NH), 133.6 (CHvCH2), 128.9, 128.8, 127.4, 116.4
(CH2vCH), 45.6 (PhCH2NH), 42.2 (CH2CHvCH2), 33.7
(SCH2); m/z (ES−) 299.0627 (M − H. C12H16N2O3PS requires
299.0624).
3
148.9, 140.8, 140.6, 119.0, 87.7 (1′-CH), 85.1 (d, JC–P 8.7,
4′-CH), 73.1 (2′-CHOH), 71.1 (3′-CHOH), 43.3 (5′-NH2CH2), 11.6
2
(d, JC–P 3.4, CH3S); m/z (ES−) 375.0643 (M − H. C11H16N6O5PS
requires 375.0646).
Kinetic studies on the decomposition of ethanolamine-
N-thiophosphoramidate 13
Buffers were prepared using CAPS (pH 10.5 and 10.17), CHES
(pH 9.81, 9.44 and 9.06), EPPS (pH 8.44 and 8.00), HEPES (pH
7.50 and 7.10), MES (pH 6.60, 6.00 and 5.88) and acetate (pH
4.80 and 4.66) systems where the pHs were adjusted by the
addition of hydrochloric acid or hydroxide solutions (see ESI†).
Crude, lyophilised ethanolamine thiophosphoramidate 13
(30 mg) was dissolved in a buffer solution (0.5 M, 4 mL or
16.
0.5 mL, see ESI†) and the mixture was lyophilised. The lyophi- The crude sample (50 mg) was dissolved in a 50 mM TEAB
lised solid was then dissolved in D2O (0.5 mL), a pH meter buffer, pH 7.5 (5 mL) and purified on DEAE Sepharose® FF
reading was taken and the mixture was transferred to a NMR column (50 mL, 10 × 3 mm, 3 mL min−1), running TEAB
tube. Owing to the fact that a rigorous deuterium exchange buffer gradient 50–200 mM. Fractions were pooled and lyophi-
was not performed, the measured pD value could not be con- lised, and the main peak in the UV trace was found to contain
verted directly to a pD value, however, for the purposes of this the desired product (87% by 31P NMR spectroscopy, 78% by
preliminary study, the uncertainty in these values (∼0.1 pD 1H NMR spectroscopy). The triethylammonium salt of the
units) was deemed acceptable. The NMR tube containing the compound was dissolved in water (5 mL) and passed through
buffered substrate was then heated to 50 °C in the NMR a Na-Dowex® 50 W × 2, 200–400 (50 mL, 30 × 2 mm, 3 mL
machine magnet, and spectra were acquired every 30 (CAPS, min−1) column, with water as the mobile phase. The fractions
CHES, EPPS, HEPES), 15 (MES), 10 (acetate buffer) or 8 (citric containing product, detected via UV trace (254 nm), were col-
buffer) minutes.
lected and lyophilised. The purity after cation exchange chromato-
The intensities of the peaks corresponding to the thiophos- graphy was estimated to be 80% by 31P NMR spectroscopy
phoramidate, normalised with the highest intensity peak in and 68% by 1H NMR spectroscopy). δH(700 MHz; D2O) 7.74
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(1 H, s, 8-H), 7.10–6.99 (5 H, m, Ar-H), 5.58 (1 H, d, J 4.5, alkylating agent was added (ESI Table 3†) along with
1′-CH), 4.47 (1 H, app t, J 5.0, 2′-CHOH), 4.17 (1 H, app t, J 5.3, additional sodium hydroxide solution (ESI Table 3†) and vigor-
3′-CHOH), 4.11–4.07 (1 H, m, 4′-CH), 3.71–3.60 (2 H, m, ous mixing was continued for either 1 h (20–33a) or overnight
CH2NH), 3.49 (1 H, dd, J 14.3 and 7.4, 5′-CH2), 3.43 (1 H, dd, J (20–33b,c). Then, ether extraction was performed (20–33a: 3 ×
14.6 and 3.4, 5′-CH2), 3.29–3.17 (2 H, m, SCH2); δP[1H] 5 mL; 20–33b,c: 3 × 20 mL) and the aqueous layer was lyophi-
(162 MHz; D2O) 22.7–22.5 (m, NHPS); δC(101 MHz; D2O) 172.9 lized. In the examples where a white precipitate appeared
3
(d, JC–P 2.6, CvO), 158.7, 153.7, 151.1, 140.2, 137.4, 128.8, during the extraction, the sample was centrifuged and the pre-
128.5, 128.2, 127.5, 127.3, 126.9, 126.7, 116.5, 87.7 (1′-CH), cipitate was dried overnight in a vacuum desiccator before
82.0 (4′-CH), 73.4 (2′-CHOH), 70.9 (3′-CHOH), 45.0 (CH2NH), being analysed. The crude material was then subjected to
2
41.4 (5′-CH2), 33.4 (d, JC–P 12.4, SCH2); m/z (ES−) 524.1127 1H and 31P NMR analyses to assess conversion levels, and
(M − H. C19H23N7O7PS requires 524.1123).
13C NMR analyses were used to confirm the identity of the
major product.
Kinetic study of the alkylation of thiophosphate ion using
5′-deoxy-5′-iodoguanosine 19
A stock solution of 100 mM NaOH with 10% D2O was made
with NaOH (0.5 mL, 1 M), H2O (4 mL) and D2O (0.5 mL).
5′-iodo 5′-deoxyguanosine (19 mg, 0.05 mmol) and tribasic
sodium thiophosphate (0.09 g, 0.5 mmol) were dissolved in
the stock NaOH solution (0.5 mL). The solution was trans-
ferred into a NMR tube and reaction progress at 50 °C was
monitored in the NMR spectrometer by 31P NMR spectroscopy
(202 MHz, 128 repetitions). Two runs were performed with
time points being taken either every 1 h or every 10 min.
Summary of spectroscopic data
20a.
δH(700 MHz; CD3OD) 8.78–8.75 (1 H, m, Ar-H), 8.20–8.17 (1 H,
m, Ar-H), 7.82 (1 H, d, J 7.0, Ar-H), 7.73–7.67 (2 H, m, Ar-H),
7.50 (1 H, br s, CF3CCHC), 7.48–7.32 (4 H, m, Ar-H) 4.56 (2 H,
d, J 11.2, CH2NH), 3.80 (2 H, d, J 8.6, SCH2); δP[1H](283 MHz;
CD3OD) 23.7–23.4 (m, NHPS); δF(376 MHz; CD3OD) −63.8
(s, CF3); δC(176 MHz; CD3OD) 149.2, 145.7, 142.2 (d, 3JC–P 10.8,
Kinetic studies of the alkylations of benzylamine-N-
thiophosphoramidate 18 and ethanolamine-N-
thiophosphoramidate 13 ion with bromoethanol and 5′-deoxy-
5′-iodoguanosine 19
3
CCH2NH), 138.0 (m, JC–P not resolved, SCH2C), 136.5, 130.9,
5′-Iodo-5′-deoxyguanosine 19 (19 mg, 0.05 mmol) or 2-bromo-
ethanol (3.5 μL, 0.05 mmol) was measured directly into an
NMR tube. Crude benzylamine-N-thiophosphoramidate
(101.5 mg) or ethanolamine-N-thiophosphoramidate (78.5 mg)
was dissolved in D2O (0.5 mL) and added to the alkylating
agent. The mixture was then subjected to 31P NMR spectro-
scopic analyses at 50 °C over a period of 12 h, with spectra
being collected every 30 minutes. All the signals appearing in
the spectra were integrated. The normalised peak area for the
signal at 25 ppm (quintet in the coupled spectra, J = 10.7 Hz),
corresponding to the alkylated product, was then plotted
against time and these data were used for kinetic fittings.
2
129.7, 128.7, 128.4, 126.9, 126.2–126.0 (m, JC–F not resolved,
Ar), 124.9 (1JC–F 272, CF3), 123.9–123.7 (m, JC–F not resolved),
3
3
122.8 (q, JC–F 3.0, Ar), 121.0, 45.6 (CH2NH), 30.0 (SCH2),
the other peaks have not been resolved; m/z (ES−) 411.0547
(M − H. C18H15N2O2F3PS requires 411.0549).
20b.
δH(700 MHz; CD3OD) 7.61 (1 H, s, Ar(CF3)), 7.52 (1 H, d, J 7.6,
Ar(CF3)), 7.46 (1 H, d, J 7.6, Ar(CF3)), 7.43 (1 H, t, J 7.6, Ar
(CF3)), 7.32 (2 H, d, J 7.6, Ar-H), 7.23 (2 H, app t, J 7.5, Ar-H),
7.15 (1 H, t, J 7.3, Ar-H), 3.91 (2 H, d, J 9.3, SCH2), 3.86 (2 H, d,
J 10.1, CH2NH); δP[1H](283 MHz; CD3OD) 22.3 (app qn, J 9.3,
NHPS); δF(376 MHz; CD3OD) −63.9 (s, CF3); δC(176 MHz;
CD3OD) 142.8–142.7 (m, Ar), 140.0–139.9 (m, Ar), 131.0, 130.0
N-Thiophosphorylation of simple hydrophobic amine library
and S-alkylation of the resulting N-thiophosphoramidate
anions 20–33a–c
Details of quantities are summarized in tabular format in the
ESI.†
An amine (ESI Table 3,† RNH2) was mixed with sodium
hydroxide solution (5 eq. of a 1 M aqueous solution: 20–33a:
0.9 mL, 0.9 mmol; 20–33b,c: 2.425 mL, 2.425 mmol) and water
(20–33a: 0.116 mL; 20–33b,c: 0.312 mL) in a round-bottomed
flask with indentations that aim to ensure turbulent mixing.
Thiophosphoryl chloride (1 eq., 20–33a: 0.18 mmol, 0.018 mL;
20–33b,c: 0.049 mL, 0.485 mmol) dissolved in THF (20–33a:
0.548 mL; 20–33b,c: 1.476 mL) was added dropwise to the
aqueous mixture over the course of 10 min. After 1 h of vigor-
ous mixing to allow N-thiophosphorylation to take place, an
2
1
(q, JC–F 31), 128.45, 128.40, 127.9, 126.3, 124.4 (q, JC–F 272,
3
3
CF3), 123.8–123.7 (m, JC–F, Ar), 122.9 (q, JC–F 3.8, Ar),
45.4 (CH2NH), 34.6 (SCH2); m/z (ES−) 360.04420
(M − H. C15H14NO2F3PS requires 360.04405).
20c.
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3
δH(700 MHz; CD3OD) 7.83 (1 H, br s, Ar-H), 7.65 (1 H, d, J 6.8, (CH2NH), 32.4 (SCH2), 24.2 (d, JC–P 6.7, CH2CH3), 12.8
Ar-H), 7.51–7.46 (2 H, m, Ar-H), 4.12 (2 H, d, J 10.1, SCH2), 2.61 (CH2CH3); m/z (ES−) 320.08799 (M − H. C16H19NO2PS requires
(2 H, dt, J 10.2 and 7.4, SCH2), 1.60 (2 H, app sx, J 7.4, 320.08796).
CH2CH3), 0.99 (3 H, t, J 7.4, CH2CH3); δP[1H](283 MHz;
CD3OD) 23.7 (app qn, J 10.2, NHPS); δF(376 MHz; CD3OD)
22a.
3
−63.9 (s, CF3); δC(176 MHz; CD3OD) 142.9 (d, JC–P 7.4,
2
CCH2NH), 131.0 (4-CH), 130.0 (q, JC–F 31.5, CCF3), 128.4
3
(5-CH), 124.3 (q, JC–F 271, CF3), 123.9 (q, JC–F 3.2, 2-CH), 122.8
3
3
(q, JC–F 3.6, 6-CH), 45.4 (CH2NH), 32.3 (SCH2), 24.2 (d, JC–P
6.8, CH2CH3), 12.7 (CH2CH3); m/z (ES−) 312.04392
(M − H. C11H14NO2F3PS requires 312.04405).
21a.
δH(700 MHz; CD3OD) 8.72 (1 H, dd, J 4.2 and 1.7, Ar-H), 8.12
(1 H, dd, J 8.3 and 1.7, Ar-H), 8.02 (1 H, d, J 8.1, Ar-H), 7.79
(1 H, d, J 7.0, Ar-H), 7.78 (1 H, d, J 7.9, Ar-H), 7.68–7.60 (2 H,
m, Ar-H), 7.37–7.31 (4 H, m, Ar-H), 7.27–7.24 (1 H, m, Ar-H),
7.18 (1 H, d, J 6.8, Ar-H), 4.57 (2 H, d, J 11.1, CH2NH), 4.18
(2 H, d, J 7.0, SCH2); δP[1H](283 MHz; CD3OD) 23.6–23.5 (m,
3
NHPS); δC(176 MHz; CD3OD) 149.4, 146.0, 138.3 (d, JC–P 3.3,
SCH2C), 136.9), 136.1 (d, JC–P 11, CCH2NH), 133.7, 131.4,
129.6, 128.5, 127.9, 127.1, 126.9, 126.0, 125.4, 125.1, 125.0,
124.9, 123.5, 43.6 (CH2NH), 32.2 (SCH2); m/z (ES−) 393.0834
(M − H. C21H18N2O2PS requires 393.0832).
22b.
δH(500 MHz; CD3OD) 8.80 (1 H, dd, J 4.2 and 1.8, Ar-H), 8.21
(1 H, dd, J 8.2 and 1.8, Ar-H), 7.81 (1 H, d, J 7.0, Ar-H), 7.74
(1 H, dd, J 8.2 and 1.3, Ar-H), 7.59–7.24 (8 H, m, Ar-H),
7.22–7.20 (2 H, m, Ar-H), 4.56 (2 H, d, J 11.2, CH2NH), 3.80
(2 H, d, J 8.2, SCH2); δP[1H](283 MHz; CD3OD) 23.8–23.6 (m,
NHPS); δC(125 MHz; CD3OD) 149.2, 145.8, 140.8, 140.1 (d, 3JC–p
10.5, CCH2NH), 139.3, 138.0 (d, 3JC–P 4.4, SCH2C), 136.6, 129.7,
128.6, 128., 127.8, 126.8, 126.4, 126.3, 126.2, 126.0,
120.8, 45.5 (CH2NH), 30.1 (SCH2); m/z (ES−) 419.0993
(M − H. C23H20N2O2PS requires 419.0988).
3
21b.
δH(700 MHz; CD3OD) 8.15 (1 H, d, J 8.5, Ar-H), 7.82 (1 H, d, J
8.0, Ar-H), 7.72 (1 H, d, J 8.1, Ar-H), 7.48–7.42 (2 H, m, Ar-H),
7.40–7.38 (1 H, m, 2-CH), 7.37–7.33 (3 H, m, Ar-H), 7.23 (2 H,
app t, J 7.6, Ar-H), 7.15 (1 H, t, J 7.4, Ar-H), 4.30 (2 H, d, J 7.4,
CH2NH), 4.30 (2 H, d, J 10.2, SCH2); δP[1H](283 MHz; CD3OD)
δH(700 MHz; CD3OD) 7.57 (2 H, d, J 7.8, Ar-H), 7.50 (2 H, d, J
7.8, Ar-H), 7.40 (2 H, t, J 7.6, Ar-H), 7.35 (2 H, d, J 7.8, Ar-H),
7.32–7.27 (3 H, m, Ar-H), 7.21 (2 H, d, J 7.5, Ar-H), 7.14 (1 H, t,
J 7.5, Ar-H), 3.91 (2 H, d, J 9.0, NCH2), 3.85 (2 H, d, J 9.6,
CH2S); δP[1H](283 MHz; CD3OD) 22.5 (app qn, J 9.3, NHPS);
3
22.4–22.2 (m, NHPS); δC(176 MHz; CD3OD) 140.2 (d, JC–p 4.0,
3
CH2S), 136.3 (d, JC–P 9.0, CH2NH), 133.9, 131.5, 128., 128.0,
127.2, 127.1, 126.3, 125.5, 125.23, 125.15, 125.0, 123.5,
43.6 (CH2NH), 34.6 (SCH2); m/z (ES−) 342.07236
(M − H. C18H17NO2PS requires 342.07231).
22c.
3
δC(176 MHz; CD3OD) 140.9, 140.5 (d, JC–p 8.5, CCH2NH),
3
139.8 (d, JC–P 5.1, CCH2S), 139.5, 128.5, 128.4, 127.9, 127.8,
126.7, 126.4, 126.4, 126.2, 45.6 (CH2NH), 34.6 (SCH2); m/z
(ES−) 368.08796 (M − H. C20H19NO2PS requires 368.08796).
21c.
δH(700 MHz; CD3OD) 8.24 (1 H, d, J 8.4, Ar-H), 7.84 (1 H, d, J
8.1, Ar-H), 7.75 (1 H, d, J 8.3, Ar-H), 7.57 (1 H, d, J 7.1, Ar-H),
δH(700 MHz; CD3OD) 7.57–7.56 (2 H, m, Ar-H), 7.53–7.51 (2 H, 7.50 (1 H, ddd, J 8.3, 6.8 and 1.3, 7-CH), 7.43 (1 H, ddd, J 8.4,
m Ar-H), 7.47–7.45 (2 H, m, Ar-H), 7.39 (1 H, t, J 7.7, Ar-H), 6.8 and 1.3, Ar-H), 7.40 (1 H, dd, J 8.1, 7.1, Ar-H), 4.51 (2 H, d, J
7.30–7.27 (1 H, m, Ar-H), 4.08 (2 H, d, J 9.8, CH2NH), 2.63 (2 H, 7.7, CH2NH), 2.64 (2 H, dt, J 10.5 and 7.4, SCH2), 1.63 (2 H,
dt, J 10.5 and 7.4, SCH2), 1.61 (2 H, app sx, J 7.4, CH2CH3), 0.95 app sx, J 7.4, CH2CH3), 0.95 (3 H, t, J 7.4, CH2CH3); δP[1H]
(3 H, t, J 7.4, CH2CH3); δP[1H](283 MHz; CD3OD) 23.8 (app qn, (283 MHz; CD3OD) 23.8–23.6 (m, NHPS); δC(176 MHz; CD3OD)
3
3
J 9.7, NHPS); δC(176 MHz; CD3OD) 140.9, 140.5 (d, JC–p 8.5, 136.3 (d, JC–P 9.0, CCH2NH), 133.9, 131.5, 128.1, 127.2, 125.5,
CCH2NH), 139.5, 128.4, 127.8, 126.7, 126.4, 126.4, 45.6 125.2, 125.1, 125.0, 123.5, 43.6 (CH2NH), 32.2 (SCH2), 24.0 (d,
2670 | Org. Biomol. Chem., 2013, 11, 2660–2675
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Paper
3JC–P 6.5, CH2CH3), 12.5 (CH2CH3); m/z (ES−) 294.07232 (CH2CH3); m/z (ES−) 382.07098 (M − H. C17H21NO3PS2 requires
(M − H. C14H17NO2PS requires 294.07231).
382.07060).
23a.
24a.
δH(700 MHz; CD3OD) 8.77–8.75 (1 H, m, Ar-H), 8.18–8.16 (1 H,
m, Ar-H), 7.76 (1 H, d, J 7.1, Ar-H), 7.68 (1 H, d, J 8.1, Ar-H),
7.52–7.48 (2 H, m, Ar-H), 7.41–7.37 (2 H, m, Ar-H), 7.24 (1 H, t,
J 7.5, Ar-H), 7.14 (1 H, t, J 7.5, Ar-H), 7.10 (1 H, t, J 7.5, Ar-H),
7.06 (1 H, t, J 7.6, Ar-H), 6.96–6.92 (2 H, m, Ar-H), 4.62 (2 H, s,
CH2OH), 4.52 (2 H, d, J 11.0, CH2NH), 3.92 (2 H, d, J 8.9,
SCH2); δP[1H](283 MHz; CD3OD) 23.9–23.8 (m, NHPS);
δC(125 MHz; CD3OD) 149.2, 145.7, 141.9, 136.6, 133.0, 132.8,
141.3 (d, 3JC–P 11.2, CCH2NH), 129.7, 129.1, 128.4, 127.9, 127.5,
127.5, 127.4), 127.3, 127.1, 120.9, 61.7 (CH2OH), 44.0 (CH2NH),
30.1 (SCH2); m/z (ES−) 481.0822 (M − H. C24H22N2O2PS
requires 481.0815).
δH(700 MHz; CD3OD) 8.66 (1 H, dd, J 4.1 and 1.7, Ar-H), 8.24
(1 H, d, J 9.2, Ar-H), 8.11 (1 H, d, J 7.5, Ar-H), 8.10 (1 H, d, J 7.6,
Ar-H), 7.99–7.89 (6 H, m, Ar-H), 7.76 (1 H, d, J 7.0, Ar-H), 7.73
(1 H, d, J 7.7, Ar-H), 7.53 (1 H, d, J 8.2, Ar-H), 7.32–7.29 (1 H,
m, Ar-H), 7.21 (1 H, dd, J 8.2 and 4.1, Ar-H), 4.58 (2 H, d,
J 11.7, CH2NH), 4.44 (2 H, d, J 7.4, SCH2); δP[1H](283 MHz;
CD3OD) 23.4–23.2 (m, NHPS); δC(125 MHz; CD3OD) 149.3,
145.9, 137.8 (d, JC–P not resolved, SCH2C), 134.0 (d, JC–P 11.2,
CCH2NH), 136.6, 131.3, 130.8, 130.5, 128.5, 129.5, 128.4, 127.0,
126.7, 126.3, 126.1, 125.4, 125.8, 124.4, 124.4, 124.2,
123.2, 120.7, 44.0 (CH2NH), 30.1 (SCH2); m/z (ES−) 467.0996
(M − H. C27H20N2O2PS requires 467.0988).
3
3
23b.
24b.
δH(700 MHz; CD3OD) 7.56 (1 H, d, J 7.7, Ar-H), 7.54 (1 H, d, J
7.7, Ar-H), 7.28–7.10 (9 H, m, Ar-H), 7.06–7.02 (2 H, m, Ar-H),
4.71 (2 H, s, CH2OH), 4.06 (2 H, d, J 9.4, CH2NH), 3.81 (2 H, d,
J 9.4, SCH2); δP[1H](283 MHz; CD3OD) 22.6 (app qn, J 9.4,
NHPS); δC(176 MHz; CD3OD) 141.9 (CCH2OH), 139.9
δH(700 MHz; CD3OD) 8.39 (1 H, d, J 9.2, Ar-H), 8.14 (1 H, t, J
7.5, Ar-H), 8.09–8.05 (1 H, m, Ar-H), 8.06 (1 H, d, J 7.7, Ar-H),
8.00 (1 H, s, Ar-H), 7.97–7.93 (1 H, m, Ar-H), 7.30 (1 H, d, J 7.3,
Ar-H), 7.18 (1 H, t, J 7.6, Ar-H), 7.12 (1 H, t, J 7.4, Ar-H), 4.56
(2 H, d, J 7.7, CH2NH), 3.89 (2 H, d, J 10.2, SCH2); δP[1H]
(283 MHz; CD3OD) 22.6–22.3 (m, NHPS); δC(176 MHz; CD3OD)
140.1 (CCH2NH), 134.2 (CH2S), 131.3, 130.8, 130.5, 128.5,
128.4, 127.9, 127.0, 126.9, 126.5, 126.4, 126.2, 125.5, 124.5,
124.4, 123.1, 43.7 (CH2NH), 34.5 (SCH2); m/z (ES−) 416.08815
(M − H. C24H19NO2PS requires 416.08796).
3
(i-C6H5CH2S), 133.0, 132.8, 131.3 (d, JC–P 8.1, CCH2NH),
128.8–126.4 (12 × s), 126.5, 61.3 (CH2OH), 46.1 (CH2NH), 35.4
(SCH2); m/z (ES−) 430.07103 (M − H. C21H21NO3PS2 requires
430.07059).
23c.
24c.
δH(700 MHz; CD3OD) 7.64 (1 H, d, J 7.7, Ar-H), 7.54 (1 H, d, J
7.7, Ar-H), 7.28 (1 H, t, J 7.5, Ar-H), 7.25 (1 H, t, J 7.5, Ar-H),
7.17 (1 H, t, J 7.5, Ar-H), 7.14 (1 H, t, J 7.5, Ar-H), 7.06 (1 H, d, J
7.7, Ar-H), 7.05 (1 H, d, J 7.7, Ar-H), 4.72 (2 H, s, CH2OH), 4.17
(2 H, d, J 9.4, CH2NH), 2.56 (2 H, dt, J 10.5 and 7.4, SCH2), 1.60
(2 H, app sx, J 7.4, CH2CH3), 0.90 (3 H, t, J 7.4, CH2CH3); δP[1H]
(283 MHz; CD3OD) 23.9 (app qn, J 9.8, NHPS); δC(176 MHz; δH(700 MHz; CD3OD) 8.39 (1 H, d, J 9.2, ArH), 8.16–8.13 (2 H,
3
CD3OD) 141.9 (CCH2OH), 133.0, 132.8, 131.3 (d, JC–P 11.2, m, Ar-H), 8.12–8.11 (2 H, m, Ar-H), 8.09 (1 H, d, J 9.2, Ar-H),
CCH2NH), 129.1, 127.9, 127.5, 127.4, 127.3, 62.3 (CH2OH), 46.1 8.01–7.99 (2 H, m, Ar-H), 7.96 (1 H, t, J 7.6, Ar-H), 4.46 (2 H, d,
3
(CH2NH), 35.4 (SCH2), 27.2 (d, JC–P 6.2, CH2CH3), 16.8 J 7.7, CH2NH), 2.63 (2 H, dt, J 9.8 and 7.4, SCH2), 1.60 (2 H,
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app sx, J 7.4, CH2CH3), 0.94 (3 H, t, J 7.4, CH2CH3); δP[1H] (3 H, d, J 12.1, NCH3), 1.75 (2 H, app sx, J 7.4, CH2CH3), 1.04
(283 MHz; CD3OD) 23.7–23.5 (m, NHPS); δC(176 MHz; CD3OD) (3 H, t, J 7.4, CH2CH3); δP[1H](283 MHz; CD3OD) 24.9–24.6 (m,
3
134.4 (d, 3JC–P 11.7, CCH2NH), 131.3, 130.8, 130.5, 128.5, 127.0, NHPS); δC(176 MHz; CD3OD) 131.5, 131.4, 129.4 (d, JC–P 9.1),
126.9, 126.5, 126.4, 125.5, 124.5, 124.4, 124.3, 44.1 (CH2NH), 128.5, 127.1, 125.3, 124.8, 124.4, 44.2 (CH2NH), 32.4 (SCH2),
3
3
32.5 (SCH2), 24.2 (d, JC–P 6.7, CH2CH3), 12.7 (CH2CH3); m/z 31.8 (NCH3), 24.7 (d, JC–P 5.5, CH2CH3), 12.7 (CH2CH3); m/z
(ES−) 368.08837 (M − H. C20H19NO2PS requires 368.08796).
(ES−) 358.10394 (M − H. C19H21NO2PS requires 358.10361).
25a.
26a.
δH(700 MHz; CD3OD) 8.79 (1 H, dd, J 4.2 and 1.8, Ar-H), 8.21
(1 H, dd, J 8.2 and 1.8, Ar-H), 7.71 (1 H, dd, J 8.2 and 1.3, Ar-
δH(700 MHz; CD3OD) 8.67 (1 H, dd, J 4.1 and 1.7, Ar-H), 8.23 H), 7.62–7.60 (1 H, m, Ar-H), 7.43 (1 H, dd, J 8.2 and 4.2, Ar-H),
7.39 (1 H, dd, J 8.1 and 7.2, Ar-H), 7.10–6.96 (8 H, m, Ar-H),
6.81–6.79 (2 H, m, Ar-H), 4.51 (1 H, J 12.7 and 9.6, SCH2),
(1 H, d, J 9.1, Ar-H), 8.08 (2 H, t, J 7.7, Ar-H), 7.99–7.89 (6 H, m,
Ar-H), 7.76 (1 H, d, J 7.0, Ar-H), 7.74 (1 H, d, J 7.7, Ar-H), 7.53
(1 H, d, J 8.1, Ar-H), 7.32–7.29 (1 H, m Ar-H), 7.20 (1 H, dd, J 4.40–4.35 (1 H, m, CH2CH), 4.31 (1 H, J 12.7 and 10.3, SCH2),
3.19 (1 H, J 13.1 and 4.6, CH2CH), 2.74 (1 H, J 13.1 and 9.5,
CH2CH); δP[1H](283 MHz; CD3OD) 21.7–21.5 (m, NHPS);
8.2 and 4.1, Ar-H), 4.59 (2 H, d, J 11.5, CH2N), 4.44 (2 H, d, J
7.4, SCH2), 3.30–3.28 (3 H, m, NCH3); δP[1H](283 MHz; CD3OD)
23.3–23.1 (m, NPS); δC(176 MHz; CD3OD) aromatic signals δC(176 MHz; CD3OD) 149.2, 149.0, 145.9, 144.1–144.0 (un-
resolved, CCHNH), 138.5, 137.8–137.7 (unresolved, SCH2C),
136.6, 136.4, 129.7–125.8 (11 × s), 120.9, 120.7, 57.9–57.6 (un-
could not be assigned owing to the level of heterogeneity of
this particular sample, 43.8 (CH2N), 38.6–37.8 (m, NCH3), 29.9
(SCH2); m/z (ES−) 457.1153 (M − H. C26H22N2O2PS requires resolved, CH2CH), 45.1–44.9 (unresolved, CH2CH), 30.1–29.8
(unresolved, SCH2); m/z (ES−) 433.1143 (M − H. C24H22N2O2PS
457.1145).
requires 433.1145).
25b.
26b.
δH(700 MHz; CD3OD) 7.19–7.03 (13 H, m, Ar-H), 6.95–6.92
(2 H, m, Ar-H), 4.46 (1 H, ddd, J 11.2, 9.2 and 5.2, CH2CH),
3.60 (1 H, J 12 and 6.9, SCH2), 3.47 (1 H, J 12 and 7.7, SCH2),
3.22 (1 H, J 13.1 and 5.2, CH2CH), 2.88 (1 H, J 13.1 and 9.0,
CH2CH); δP[1H](283 MHz; CD3OD) 20.9–20.8 (m, NHPS);
δH(700 MHz; CD3OD) 8.60 (2 H, d, J 8.8, Ar-H), 8.40 (1 H, s, Ar-
H), 7.97 (2 H, d, J 8.5, Ar-H), 7.52 (2 H, d, J 7.5, Ar-H),
7.49–7.45 (2 H, m, Ar-H), 7.43–7.40 (2 H, m, Ar-H), 7.33 (2 H, t,
J 7.6, Ar-H), 7.26 (1 H, t, J 7.4, Ar-H), 5.01 (2 H, d, J 3.8,
CH2NH), 4.15 (2 H, d, J 10.1, SCH2), 2.21 (3 H, d, J 12.2, NCH3);
δP[1H](283 MHz; CD3OD) 23.7–23.4 (m, NHPS); δC(176 MHz;
CD3OD) 140.9 (d, JC–p 5.4), 131.7, 131.6, 129.7 (d, JC–P 10.7),
128.7, 128.5, 127.6, 127.3, 126.3, 125.5, 124., 124.7,
44.5 (CH2NH), 34.3 (SCH2), 32.4 (NCH3); m/z (ES−) 406.10419
(M − H. C23H21NO2PS requires 406.10361).
3
δC(176 MHz; CD3OD) 144.4 (d, JC–P 3.7, CCHNH), 139.2 (d,
3JC–P 7.8, SCH2C), 138.5, 129.5–125.5 (6 × s), 57.7 (CH2CH),
45.7–45.4 (unresolved, CH2CH), 34.5–34.3 (unresolved, SCH2);
3
3
m/z (ES−) 382.10338 (M
−
H. C21H21NO2PS requires
382.10361).
26c.
25c.
δH(700 MHz; CD3OD) 7.18–7.03 (8 H, m, Ar-H), 6.97–6.94 (2H,
m, Ar-H), 4.45 (1 H, ddd, J 14.0, 11.2 and 5.6, CH2CH), 3.22
(1 H, J 13.2 and 5.3, CH2CH), 2.93 (1 H, CH2CH), 2.37–2.22 (m,
δH(700 MHz; CD3OD) 8.74 (2 H, d, J 8.9, Ar-H), 8.42 (1 H, s, SCH2), 1.42–1.32 (2 H, m, CH2CH3), 0.79 (3 H, t, J 7.4, CH3);
Ar-H), 7.99 (2 H, d, J 8.4, Ar-H), 7.50 (2 H, 2 × t, J 7.4, 2- and δP[1H](283 MHz; CD3OD) 22.4–22.3 (m, NHPS); δC(176 MHz;
3
7-CH), 7.51–7.48 (2 H, m, Ar-H), 7.44–7.41 (2 H, m, Ar-H) 5.17 CD3OD) 144.4 (d, JC–P not resolved, CCHNH), 138.5, 129.4,
3
(2 H, d, J 3.7, CH2NH), 2.85 (2 H, dt, J 9.8 and 7.4, SCH2), 2.30 127.4 (2 × s), 126.8, 126.0, 125.5, 57.6 (CH2CH), 45.5 (d, JC–P
2672 | Org. Biomol. Chem., 2013, 11, 2660–2675
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3
5.6, CH2CH), 32.0 (SCH2), 23.6 (d, JC–P 7.4, CH2CH3), 12.5
29b.
(CH3); m/z (ES−) 334.10399 (M − H. C17H21NO2PS requires
335.10361).
27a.
δH(700 MHz; CD3OD) 7.38 (2 H, d, J 7.3, Ar-H), 7.28 (2 H, t, J
7.6, Ar-H), 7.21 (1 H, t, J 7.3, Ar-H), 3.89 (2 H, d, J 9.9, SCH2),
2.68 (2 H, dt, J 8.7 and 7.4, CH2NH), 1.39 (2 H, app sx, J 7.4,
CH3CH2), 0.86 (3 H, t, J 7.4, CH3CH2); δP[1H](283 MHz;
CD3OD) 23.1 (app qn, J 9.5, NHPS); δC(176 MHz; CD3OD) 140.2
δH(700 MHz; CD3OD) 8.80 (1 H, dd, J 4.2 and 1.7, Ar-H), 8.25
(1 H, dd, J 8.2 and 1.7, Ar-H), 7.82 (1 H, d, J 7.1, Ar-H), 7.76
(1 H, d, J 8.1, Ar-H), 7.49–7.42 (2 H, m, Ar-H), 7.20–7.11 (5 H,
m, Ar-H), 4.55 (2 H, d, J 11.2, SCH2), 3.74 (2 H, d, J 7.7,
CH2NH); δP[1H](283 MHz; CD3OD) 23.7–23.5 (m, NHPS);
3
(d, JC–P 6.1, SCH2C), 128.7, 128.2, 126.5, 43.8 (CH2NH), 34.6
(d, 2JC–P 2.8, SCH2), 24.5 (d, 3JC–P 8.8, CH3CH2), 10.7 (CH3CH2);
m/z (ES−) 244.05622 (M
−
H. C10H15NO2PS requires
244.05666).
30a.
3
δC(176 MHz; CD3OD) 149.2, 145.8, 141.0–140.8 (m, JC–P not
3
resolved, CCH2NH), 138.2–138.0 (m, JC–P not resolved,
SCH2C), 136.6, 129.7, 128.6, 127.7, 127.2, 126.9, 126.1 (2 × s),
120.9, 45.8 (CH2NH), 29.8 (SCH2); m/z (ES−) 343.0678
(M − H. C17H16N2O2PS requires 343.0678).
28a.
δH(700 MHz; CD3OD) 8.81 (1 H, dd, J 4.2 and 1.6, Ar-H), 8.37 (1
H, d, J 4.3, Ar-H), 8.23 (1 H, dd, J 8.2 and 1.4, Ar-H), 7.82 (1 H,
d, J 7.0, Ar-H), 7.74 (1 H, d, J 8.0, Ar-H), 7.65 (1 H, td, J 7.7 and
1.7, Ar-H), 7.47–7.42 (2 H, m, Ar-H), 7.20 (1 H, d, J 7.8, Ar-H),
7.17 (1 H, dd, J 6.9 and 5.5, Ar-H), 4.50 (2 H, d, J 10.4, SCH2),
3.05–2.99 (2 H, m, CH2CH2NH), 2.81 (2 H, t,
J 7.2,
CH2CH2NH); δP[1H](283 MHz; CD3OD) 23.6 (app qn, J 10.1,
NHPS); δC(176 MHz; CD3OD) 160.0, 149.2, 148.2, 145.8,
137.9–137.8 (m, 3JC–P not resolved, SCH2C), 137.0, 136.6, 129.7,
128.5, 126.9, 126.1, 123.6, 121.5, 120.9, 41.6 (CH2NH), 39.1
δH(500 MHz; CD3OD) 8.89 (1 H, dd, J 4.2 and 1.8, Ar-H), 8.30
(1 H, dd, J 8.3 and 1.7, Ar-H), 7.90–7.86 (1 H, m, Ar-H), 7.82
(1 H, dd, J 8.2 and 1.3, Ar-H), 7.56–7.49 (2 H, m, Ar-H),
5.83–5.72 (1 H, m, CH2vCH), 4.99 (1 H, dq, J 17.1 and 1.7,
CHHvCH), 4.91–4.87 (1H, m, CHHvCH), 4.57 (2 H, d, J 11.1,
SCH2), 3.23 (2 H, ddt, J 8.8, 5.7 and 1.5, CH2NH); δP[1H]
(283 MHz; CD3OD) 23.8–23.6 (m, NHPS); δC(125 MHz; CD3OD)
3
(d, JC–P 7.6, CH2CH2NH), 30.2 (SCH2); m/z (ES−) 358.0786
(M − H. C17H17N3O2PS requires 358.0784).
30c.
3
3
149.5, 146.0, 138.3 (d, JC–P 4.6, SCH2C), 137.5 (d, JC–P 9.9,
CHCH2), 136.9, 130.0, 128.9, 127.2, 126.4, 121.2, 113.7
2
(CH2vCH), 44.8 (CH2NH), 30.0 (d, JC–P 2.7, SCH2); m/z (ES−)
293.0522 (M − H. C13H14N2O2PS requires 293.0519).
29a.
δH(700 MHz; CD3OD) 8.42 (1 H, ddd, J 5.0, 1.7 and 0.9, Ar-H),
7.74 (1 H, td, J 7.7 and 1.8, Ar-H), 7.37 (1 H, d, J 7.8, Ar-H), 7.24
(1 H, ddd, J 7.5, 5.0 and 1.0, Ar-H), 3.22 (2 H, dt, J 9.8 and 7.1,
CH2CH2NH), 2.98 (2 H, t, J 7.1, CH2CH2NH), 2.55 (2 H, dt, J
10.3 and 7.3, SCH2), 1.58 (2 H, app sx, J 7.4, CH2CH3), 0.93 (3
H, t, J 7.4, CH2CH3); δP[1H](283 MHz; CD3OD) 24.0 (app qn, J
10.1, NHPS); δC(176 MHz; CD3OD) 159.9, 148.2, 137.1, 123.8,
3
121.5, 41.6 (CH2CH2NH), 39.2 (d, JC–P 8.0, CH2CH2NH), 32.1
δH(500 MHz; CD3OD) 8.89 (1 H, dd, J 4.2 and 1.8, Ar-H), 8.30
(1 H, dd, J 8.3 and 1.7, Ar-H), 7.89 (1 H, dd, J 7.1 and 1.0, Ar-
H), 7.82 (1 H, dd, J 8.2 and 1.2, Ar-H), 7.56–7.49 (2H, m, Ar-H),
4.56 (2 H, d, J 11.1, SCH2), 2.57 (2 H, dt, J 8.7 and 7.4, CH2NH),
1.30 (2 H, app sx, J 7.4, CH3CH2), 0.77 (3 H, t, J 7.4, CH3CH2);
δP[1H](283 MHz; CD3OD) 24.2–24.0 (m, NHPS); δC(125 MHz;
3
(SCH2), 23.9 (d, JC–P 6.4, CH2CH3), 12.5 (CH2CH3); m/z (ES−)
259.06778 (M − H. C10H16N2O2PS requires 259.06756).
31a.
3
CD3OD) 149.5, 146.1, 138.3 (d, JC–P 4.6, SCH2C), 136.9, 129.9,
2
128.8, 127.2, 126.4, 121.2, 43.8 (CH2NH), 30.1 (d, JC–P 2.7,
SCH2), 24.4 (d, JC–P 9.1, CH3CH2), 10.7 (CH3CH2); m/z (ES−)
2
295.0677 (M − H. C13H16N2O2PS requires 295.0675).
δH(700 MHz; CD3OD) 8.83 (1 H, dd, J 4.0 and 1.3, Ar-H),
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Organic & Biomolecular Chemistry
8.27–8.24 (1 H, m, Ar-H), 7.88 (1 H, d, J 7.0, Ar-H), 7.76 (1 H, d, t, J 7.4, CH2CH3); δP[1H](283 MHz; CD3OD) 24.2 (app qn, J 9.9,
J 8.0, Ar-H), 7.57 (1 H, d, J 7.6, Ar-H), 7.51–7.43 (2 H, m, Ar-H), NHPS); δC(176 MHz; CD3OD) details for major conformer
7.01–6.98 (1 H, m, Ar-H), 6.95–6.91 (2 H, m, Ar-H), 4.64–4.61 66.3 (O(CH2)2), 56.8 (NCH2), 53.4 ((CH2)2N), 40.2 (CH2NH),
(2 H, m, SCH2), 4.30–4.25 (1 H, m, CHNH), 2.70–2.55 (2 H, m, 32.1 (SCH2), 27.8–27.4 (m, CH2CH2CH2), 24.2–23.8
3-CH2), 1.95–1.87 (1 H, m, CHH), 1.79–1.71 (1 H, m, CHH), (m, CH2CH3), 12.7–12.5 (m, CH2CH3); m/z (ES−) 281.10967
1.68–1.55 (2 H, m, CH2); δP[1H](283 MHz; CD3OD) 21.3 (app q, (M − H. C10H22N2O3PS requires 281.10943).
J 10.3, NHPS); δC(176 MHz; CD3OD) 149.2, 146.0, 140.0–139.8
33a.
3
3
(m, JC–P not resolved, CCHNH), 138.0–137.9 (m, JC–P not
resolved, SCH2C), 136.8, 136.5, 129.7, 128.9, 128.6, 128.0,
126.9, 126.2, 125.9, 125.2, 120.9, 49.7 (CHNH), 31.9, 30.3
3
(d, JC–P 2.6), 29.0 (SCH2), 19.7; m/z (ES−) 383.0987
(M − H. C20H20N2O2PS requires 383.0988).
31c.
δH(700 MHz; CD3OD) 8.79 (1 H, dd, J 4.2 and 1.7, Ar-H), 8.23
(1 H, dd, J 8.2 and 1.7, Ar-H), 7.81 (1 H, d, J 7.0, Ar-H), 7.74
(1 H, d, J 8.2, Ar-H), 7.47–7.44 (1 H, m, Ar-H), 7.42 (1 H, dd,
J 8.2 and 4.2, Ar-H), 7.16–7.12 (2 H, m, Ar-H), 6.98 (2 H, t, J 8.8,
Ar-H), 4.54 (2 H, d, J 11.1, CH2NH), 3.73 (2 H, d, J 8.3, SCH2);
δP[1H](283 MHz; CD3OD) 23.6–23.4 (m, NHPS); δF(376 MHz;
CD3OD) −(118.8–119.0) (m, Ar-F); δC(176 MHz; CD3OD) 162.4,
149.2, 145.8, 138.1–137.9 (m, PSCH2C), 137.0–136.8 (m,
δH(700 MHz; CD3OD) 7.63 (1 H, d, J 7.2, Ar-H), 7.09–7.03 (2 H,
m, Ar-H), 6.99 (1 H, d, J 7.3, Ar-H), 4.34–4.29 (1 H, m, CHNH),
2.80–2.64 (4 H, m, 3-CH2 and SCH2), 2.11–2.04 (1 H, m, 4-CH2),
1.96–1.89 (1 H, m, 4-CH2), 1.89–1.82 (1 H, m, 2-CH2), 1.78–1.62
(4 H, m, 2-CH2 and CH2CH3), 0.99 (3 H, t, J 7.4, CH2CH3);
δP[1H](283 MHz; CD3OD) 22.2–22.0 (m, NHPS); δC(176 MHz;
CD3OD) 140.1–140.0 (m, CCHNH), 136.7, 128.9, 128.1, 126.0,
125.2, 49.7 (CHNH), 32.5 (2-CH), 32.3 (SCH2), 29.0 (4-CH), 23.9
3
CCH2NHP), 136.6, 129.7, 129.0 (d, JC–F, 8.0), 128.6, 126.9,
2
126.1, 121.0, 114.2 (d, JC–F, 21.4), 45.0 (CH2NH), 29.8 (SCH2);
m/z (ES−) 361.0583 (M − H. C17H15N2O2FPS requires 361.0581).
33c.
3
(d, JC–P 7.0, CH2CH3), 19.8 (3-CH), 12.6 (CH2CH3); m/z (ES−)
284.08826 (M − H. C13H19NO2PS requires).
32a.
δH(700 MHz; CD3OD) 7.41–4.37 (2 H, m, Ar-H), 7.00–6.95 (2 H,
m, Ar-H), 4.02 (2 H, d, J 9.4, CH2NH), 2.61 (2 H, dt, J 10.3 and
7.3, SCH2), 1.61 (2 H, app sx, J 7.4, CH2CH3), 0.95 (3 H, t, J 7.4,
CH2CH3); δP[1H](283 MHz; CD3OD) 23.8 (app qn, J 9.8 NHPS);
δF(376 MHz; CD3OD) −(118.9–119.1) (m, Ar-F); δC(176 MHz;
δH(700 MHz; CD3OD) 8.86 (1 H, dd, J 4.2 and 1.7, Ar-H), 8.26
(1 H, dd, J 8.2 and 1.7, Ar-H), 7.87 (1 H, d, J 7.0, Ar-H), 7.78
(1 H, d, J 8.2, Ar-H), 7.52–7.46 (2 H, m, Ar-H), 4.54 (2 H, d, J
10.8, SCH2), 3.60 (4 H, t, J 4.6, O(CH2)2), 2.67 (2 H, dt, J 9.3 and
7.0, PNHCH2), 2.30 (4 H, br s, (CH2)2N), 2.24–2.20 (2 H, m,
NCH2), 1.50–1.44 (2 H, m, CH2CH2CH2); δP[1H](283 MHz;
CD3OD) 23.8 (app qn, J 10.1, NHPS); δC(176 MHz; CD3OD)
149.2, 145.9, 138.2–138.0 (m, SCH2C), 136.6, 129.6, 128.5,
126.9, 126.1, 121.0, 66.2 (O(CH2)2), 56.6 (NCH2), 53.3
3
CD3OD) 161.8 (d, JC–F 242.5, FC), 137.4 (d, JC–P 8.0, CCH2NH),
3
2
129.0 (d, JC–F, 8.0, 3-CH and 5-CH), 114.2 (d, JC–F, 21.5, 2-CH
3
and 6-CH), 45.0 (CH2NH), 32.1 (SCH2), 24.0 (d, JC–P
6.4, CH2CH3), 12.5 (CH2CH3); m/z (ES−) 262.04744
(M − H. C10H14NO2FPS requires 262.04724).
34.
3
((CH2)2N), 40.2 (CH2NH), 29.9 (SCH2), 27.3 (d, JC–P 7.5,
CH2CH2NHP); m/z (ES−) 380.1202 (M − H. C17H23N3O3PS
requires 380.1203).
32c.
δH(500 MHz; CD3OD) 8.87–8.84 (1 H, m, Ar-H), 8.29–8.25 (1 H,
m, Ar-H), 8.00–7.96 (1 H, m, Ar-H), 7.77–7.74 (1 H, m, Ar-H),
7.53–7.44 (2 H, m, Ar-H), 4.66–4.62 (2 H, m, SCH2); δP[1H]
3
δH(700 MHz; CD3OD) 3.70–3.66 (4 H, m, O(CH2)2), 2.91 (2 H, (283 MHz; CD3OD) 18.1 (t, JH–P 6.9, NHPS); δC(125 MHz;
3
dt, J 9.6 and 6.8, SCH2), 2.63 (2 H, dt, J 10.3 and 7.3, CH2NH), CD3OD) 149.1, 146.1, 138.8 (d, JC–P 7.0, SCH2C), 136.7, 130.1,
2.46 (4 H, br s, (CH2)2N), 2.44–2.40 (2 H, m, NCH2), 1.75–1.65 128.5, 126.4, 126.3, 120.7, 43.4 (CH2NH); m/z (ES−) 254.0047
(2 H, m, CH2CH2CH2), 1.65–1.60 (2 H, m, CH2CH3), 0.97 (3 H, (M − H. C10H9NO2PS requires 254.0046).
2674 | Org. Biomol. Chem., 2013, 11, 2660–2675
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December 2012.
Acknowledgements
We thank Durham University (MT), EPSRC (FLC), Wolfson
Small Grants Scheme (SLC/PWD) and the Royal Society 16 M. Lasker, C. D. Bui, P. G. Besant, K. Sugawara, P. Thai,
(DRWH) for funding. In addition, we are grateful to Drs Alan
Kenwright and Ian Cummins for NMR and MS support.
G. Medzihradszky and C. W. Turck, Protein Sci., 1999, 8,
2177–2185.
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T. Fraczyk, J. Ciesla, A. Les, Z. Szewczuk and W. Rode,
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