Insight into the mechanism of three component condensation leading to aminomethylenebisphosphonates

Article abstract of DOI:10.1016/j.jorganchem.2009.07.025

Three-component reaction of a primary amine, diethyl phosphite and triethyl orthoformate followed by acid hydrolysis of the adduct provides N-substituted aminomethylenebisphosphonic acids in good yields. Being extremely versatile, it is commonly utilized for preparation of compounds possessing potential antiosteoporotic, antibacterial, anticancer, antiparasitic or herbicidal activity. However, the mechanism of the reaction remains unknown. p-Nitroaniline has been found an interesting tool to shed light on this matter. Its use allowed to separate and identify four intermediates, both non-phosphorus and phosphorus containing, and subsequently suggest the mechanism of the whole process. The acquired knowledge was helpful in explanation the route and the final product structure obtained for more complex reaction proceeding with the use of 4-aminopyridine. Additional alkylation of the pyridine nitrogen atom, leading to unexpected N-(1-alkylpyridinium-4-amino)methylenebisphosphonic acids was unambiguously proved.

Full text of DOI:10.1016/j.jorganchem.2009.07.025

Journal of Organometallic Chemistry 694 (2009) 3806–3813
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Insight into the mechanism of three component condensation leading
to aminomethylenebisphosphonates
a
a
a
b
c
´
Ewa Da˛browska , Agnieszka Burzynska , Artur Mucha , Ewa Matczak-Jon , Wanda Sawka-Dobrowolska ,
Łukasz Berlicki a, Paweł Kafarski a,
*
a
_
´
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
b
_
Department of Inorganic and Structural Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland
c Department of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2009
Received in revised form 7 July 2009
Accepted 10 July 2009
Available online 16 July 2009
Three-component reaction of a primary amine, diethyl phosphite and triethyl orthoformate followed by
acid hydrolysis of the adduct provides N-substituted aminomethylenebisphosphonic acids in good yields.
Being extremely versatile, it is commonly utilized for preparation of compounds possessing potential
antiosteoporotic, antibacterial, anticancer, antiparasitic or herbicidal activity. However, the mechanism
of the reaction remains unknown. p-Nitroaniline has been found an interesting tool to shed light on this
matter. Its use allowed to separate and identify four intermediates, both non-phosphorus and phospho-
rus containing, and subsequently suggest the mechanism of the whole process. The acquired knowledge
was helpful in explanation the route and the final product structure obtained for more complex reaction
proceeding with the use of 4-aminopyridine. Additional alkylation of the pyridine nitrogen atom, leading
to unexpected N-(1-alkylpyridinium-4-amino)methylenebisphosphonic acids was unambiguously
proved.
The contribution is dedicated to Prof. Roman
Tyka on his 85th anniversary
Keywords:
Aminomethylenebisphosphonates
Reaction mechanism
Organophosphorus chemistry
Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction
The simplest procedure for preparation of amino-
methylenebisphosphonates relies on three-component reaction
Bisphosphonic acids are hydrolytically stable analogues of pyro-
phosphate characterized by a common P–C–P fragment, in which
the oxygen-to-phosphorus bonds are replaced by the carbon-to-
phosphorus bonds. Bisphosphonates have been employed as ther-
apeutic agents for treatment of bone disorders, hypocalcaemia of
malignancy and osteoporosis for over two decades [1]. Despite this,
the molecular mode of their action is still not clear and attracts
considerable attention [2]. Recently, a promising drug delivery sys-
tem using bisphosphonate moiety for targeting osseous tissues
was also proposed [3]. Additionally, bisphosphonates have been
found to have antibacterial [4] and anticancer [5] properties and
between a primary or secondary amine, triethyl orthoformate
and diethyl phosphite, followed by acid hydrolysis (Scheme 1)
[10]. Although this straightforward procedure affords desired
products of a wide structural variety, the detailed route leading
to the target structure is not recognized yet. The yield is usually
satisfactory, however it happens unpredictably low. Moreover,
unexpected by-products are obtained in those particular cases.
In this paper we wish to present the results of our studies on
elucidation of the mechanism of the title three component conden-
sation. The overall reaction route has been proposed on the basis of
the structure of isolated intermediates and conversion reactions
observed between them. These findings supported elucidation of
the structure of unusual alkylated products obtained upon forma-
tion of pyridinylaminomethylenebisphosphonates.
to stimulate cd T cells of immune system, drawing interest in can-
cer immunotherapy [6].
A subclass of bisphosphonates, derivatives of aminomethylene-
bisphosphonic acid, has been also described to exhibit promising
antiparasitic [7] and herbicidal activities [8]. It is worth to note that
a representative of the latest generation of antiosteoporotic drugs
– cycloheptylaminomethylenebisphosphonate, discovered in the
early 90s [9] and commercialized as Incadronate, belongs to such
modified bisphosphonic acids.
2. Results and discussion
2.1. Reaction with p-nitroaniline and identification of intermediates
The analysis of the general mechanism of amino-
methylenebisphosphonates formation in the condensation of dial-
kyl phosphite, trialkyl orthoformate and amine was performed using
p-nitroaniline as the nucleophilic component. This compound was
* Corresponding author. Tel.: +48 71 320 36 82; fax: +48 71 328 40 64.
E-mail address: pawel.kafarski@pwr.wroc.pl (P. Kafarski).
0022-328X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2009.07.025

E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
3807
Shortening of the reaction time to 1.5 h not only allowed to ob-
tain different ratios of the products but enabled to isolate and char-
acterize individual components of the reaction mixture (Scheme
2). Thus, the major fraction of the main product 2 (62%) precipi-
tated directly from the reaction mixture upon cooling, and it was
obtained by filtration. The residual solution contained unreacted
phosphite (68% of the filtrate, by 31P NMR) and three phosphonate
products 1–3 (1.5%, 26% and 4.5% of the filtrate, respectively). They
were separated by column chromatography, characterized and
identified, together with two non-phosphorus imine type deriva-
tives: N,N0-di(p-nitrophenyl)formamidine 4 and ethyl N-(p-nitro-
phenyl)formimidate 5. Dedicated synthetic procedures were
additionally established (for the details see the Section 3) to obtain
preparative quantities of 3–5 used in further study of the interme-
diates reactivity towards other components of the reaction mixture
(Scheme 3).
Reaction of p-nitroaniline with triethyl orthoformate, depend-
ing on the molar ratio of the two substrates provided either com-
pound 4 or 5 as the major product with satisfactory yield. As
formimidate 5 is thermally unstable the reaction had to be stopped
after 30 min. Its further subjection to elevated temperature in-
duced decomposition partially leading to compound 4. Conversion
of 5 into 4 could be also achieved in preparative manner upon ac-
tion of p-nitroaniline. When each of both compounds (4 or 5) was
reacted with diethyl phosphite, the product of the addition reac-
tion was obtained (3 and 2, respectively). Finally, phosphonates 2
and 3 reacted with excess of diethyl phosphite and yielded bis-
phosphonate 1. Similarly to 4 and 5, conversion of phosphonate
2–3 could be performed by reaction of the ethoxy derivative 2 with
p-nitroaniline or, to some extend, by its thermal decomposition.
Thus, the reaction is quite complex and relies on the kinetics of for-
mation and thermodynamic stability of all the intermediates. This
also shows that the optimization of the reaction conditions can be
difficult and dependent on the structure of individual amino com-
ponent since the ratio of formation of all intermediates is deter-
mined by its reactivity.
OEt
OEt
O
P
OEt
Δ
+
+
R-NH2
OEt
EtO
H
O
P
O
P
H
N
OEt
OEt
H
OH
OH
HClaq
N
R
R
OEt
OEt
OH
OH
P
P
O
O
Scheme 1. General reaction for the synthesis of derivatives of aminomethylene-
bisphosphonic acids.
chosen intentionally because of its characteristic features advanta-
geous for this purpose. First, due to its relatively poor nucleophilic-
ity the reaction proceeded slowly enough to detect and analyze
intermediate products. Second, separation and purification pro-
cesses (crystallization and chromatography) were greatly facili-
tated as compounds containing p-nitroaniline fragment are
yellow colored, easily crystallizing solids. These properties were
also used to obtain appropriate monocrystals and to analyze their
molecular structure by X-ray diffraction.
Standard reaction condition applied for the bisphosphonate
synthesis involved 12 h of heating of three substrates under reflux.
As the result, the formation of three organophosphorus compounds
was observed (Scheme 2). Major portion of the target tetraethyl
bisphosphonate 1 crystallized from the reaction mixture. This rep-
resented 56% of the total yield. The filtrate contained not reacted
diethyl phosphite (50% relative yields obtained from 31P NMR spec-
tra of the residual liquid), non-separated amounts of the desired
compound 1 (7%), and two other organophosphorus moieties.
On the basis of further studies they were identified as diethyl
ethoxy(p-nitrophenylamino)methanephosphonate
2 (33%) and
diethyl di(p-nitrophenylamino)methanephosphonate 3 (10%). Esti-
mated overall yields of all three organophosphorus products, cal-
culated by summarizing the solid and the liquid fraction contents
are given in Scheme 2.
To rationalize the optimization process, behavior of formimi-
date 5 in the reaction with increasing excess of diethyl phosphite
H
H
N
H
N
P(O)(OEt)2
P(O)(OEt)2
N
P(O)(OEt)2
+
+
P(O)(OEt)2
OEt
2, 14.5%
HN
O2N
O2N
O2N
1, 59%
3, 4.5%
NO2
HP(O)(OEt)2
HC(OEt)3
reflux, 12 h
NH2
O2N
HP(O)(OEt)2
HC(OEt)3
reflux, 1.5 h
H
N
H
N
+
1-3
+
OEt
N
O2N
O2N
NO2
4
5
Scheme 2. Structures of phosphorus containing products (1–3) obtained in the reaction of p-nitroaniline with ethyl orthoformate and diethyl phosphite in standard
conditions (1:1:2 molar ratio of the reagents, reflux, 12 h) and non-phosphorus intermediates (4 and 5) identified in the reaction mixture when stopped after 1.5 h of the
reaction.

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E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
NH2
O2N
HC(OEt)3
(1:5); 0.5 h
HC(OEt)3
(2:1); 8 h
87%
78%
H
N
H
NH2
N
N
O2N
O2N
OEt
O2N
NO2
4
5
74%
HP(O)(OEt)2
(1:3); 6 h
HP(O)(OEt)2
(1:1); 2 h
80% by NMR
90% by NMR
NH2
; 5 h
73% by NMR
H
N
O2N
P(O)(OEt)2
NO2
H
N
P(O)(OEt)2
OEt
HN
O2N
or 31% by NMR
(4.5 h, heating only
in toluene)
O2N
3
2
70% by NMR 63% by NMR
HP(O)(OEt)2
(1:3); 4.5 h
HP(O)(OEt)2
(1:3); 8 h
H
N
P(O)(OEt)2
P(O)(OEt)2
O2N
1
Scheme 3. General reaction scheme with conditions of selective transformations between non-phosphorus intermediates (4 and 5) and phosphorus containing products (1–
3) (reactions carried out at 90 °C, in toluene or o-xylene when needed for solubility, the ratio of substrate to reagent indicated in parentheses).
was studied in some more details (Table 1). Similarly as stated be-
fore, ethoxyphosphonate 2 appeared overwhelming product at the
early stages of the reaction (<4 h) when substrates in 1:1 molar ra-
tio were heated together. Along the time 2 was slowly consumed
being converted equally into bisphosphonate and bisamino deriv-
atives (1 and 3), however, still remaining the major component
of the mixture after 8 h of the reaction. Addition of greater excess
of diethyl phosphite (3 eq.) and increasing the reaction time caused
Table 1
Monitoring the course of the reaction of formimidate 5 with increasing amount of diethyl phosphite (as relative ratio of phosphonates 1–3 calculated from 31P NMR spectra).
H
HP(O)(OEt)2
N
(from 1:1 or 1:3)
OEt
90oC
5
O2N
H
N
H
N
H
N
P(O)(OEt)2
P(O)(OEt)2
P(O)(OEt)2
+
+
P(O)(OEt)2
OEt
HN
O2N
O2N
O2N
1
2
3
NO2
Reaction time (h)
Diethyl phosphite
Ratio of the products (by 31P NMR) (%)
1
2
3
0.5
2
4
8
0.5
4
1 eq.
1 eq.
1 eq.
1 eq.
3 eq.
3 eq.
3 eq.
7
8
11
19
6
79
80
76
55
48
0
14
12
13
26
46
77
31
23
69
8
0

E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
3809
OEt
OEt
H
N
NH2
HC
EtO
OEt
OEt
R
- EtOH
- 2 EtOH
EtO
R
RNH2
Imine
- EtOH
derivatives
formation
H
N
H
R
N
R
N
EtO
R
H
P
O
OEt
Nucleophilic addition of
diethyl phosphite
to imine derivatives
H
N
H
P(O)(OEt)2
R
RNH2
N
R
P(O)(OEt)2
OEt
R
Fig. 1. The molecular structure and atom-numbering scheme for compound 3.
Displacement ellipsoids are shown at 45% probability level. Disorder of ethyl
phosphonate groups is shown.
HN
Thermal elimination
- EtOH
- RNH2
visible enhancement of the final product yield while 2 disappeared
immediately. However, the obvious conclusion that the increase in
the yield of target bisphosphonate should be achieved by increase
of molar ratio of diethyl phosphate used is not so apparent when
considering that this usually causes substantial difficulties in sep-
aration of pure final products.
N
P(O)(OEt)2
R
6
Formation of compound 3 was unambiguously shown by X-ray
studies. The molecular structure and atom numbering of 3 is given
in Fig. 1. We have found that O1–CH2–CH3 and O3–CH2–CH3 moi-
eties are disordered into two position and their two positions (O1/
O10, C2/C20 and O3/O30, C4/C40) were refined with s.o.f. = 0.56, 0.44
and 0.52, 0.48, respectively. This behavior of phosphonate ester
group was reported earlier and thus is not surprising [11].
H
P
O
EtO
OEt
H
N
P(O)(OEt)2
P(O)(OEt)2
R
Scheme 4. Suggested general mechanism of aminomethylenebisphosphonate
formation.
2.2. Reaction mechanism
Taking into consideration all the observation and accounts gi-
ven above, a mechanism of the whole process of bisphosphonate
formation could be proposed (Scheme 4). Its initial steps seem to
be similar to the Kabachnik-Fields reaction in which a carbonyl
component is replaced by triethyl orthoformate of corresponding
electrophilic character. Imines are commonly suggested as inter-
mediates of the Kabachnik-Fields reaction. Here, formation of ethyl
N-(p-nitrophenyl)formimidate 5 and N,N0-di(p-nitrophenyl)form-
amidine 4 has been clearly evidenced. Respectively, the com-
pounds are the products of single or double nucleophilic
substitution of p-nitroaniline associated with ethanol elimination
taking place on the electrophilic carbon of orthoformate. Both
imine type intermediates can easily undergo nucleophilic addition
of diethyl phosphite yielding secondary intermediate phospho-
nates. The following steps are not fully clear. We suggest a slow
thermal elimination of ethanol or an amine leading to common
hypothetical iminephosphonate 6. It has not been separated nor
spectroscopically identified, however appearance of trace phos-
phorus containing impurities has been observed during the study
of the reaction course. Structure 6 seems to represent a logical sub-
strate for subsequent hydrophosphonylation linking together two
separate synthetic pathways. Sequential substitution in the phos-
phonate system leading to the thermodynamically most stable tar-
get compound can represent an alternative option for the last step
of the reaction.
The mechanism described above can be additionally influenced
by other substrate structural or reactivity factors complicating fur-
thermore aminomethylenebisphosphonate formation. The use of
aminopyridine derivatives as nucleophilic components of the con-
densation can represent such an example. The discussion on the
reactions that do not proceed in a typical way is greatly facilitated
by the general knowledge presented above.
2.3. Alkylation of the pyridyl nitrogen atom
A series of N-pyridylaminomethylenebisphosphonates, deriva-
tives of structurally variable aminopyridines chosen as substrates,
were obtained in order to study their herbicidal activity [8]. Most
of these products had been already described in the literature
[8a,12]. The reactions with 2-amino- and 3-aminopyridine affor-
ded, as expected, the desired products 7 and 8 with satisfactory
yields, whereas in the reaction with 4-aminopyridine, surprisingly
and in contrast to literature, the product possessing additional
ethyl group was obtained. Similarly alkylated pyridinebisphospho-
nates were also identified as single products of the condensation
proceeding in trimethyl orthoformate/dimethyl phosphite and
tri(n-butyl) orthoformate/di(n-butyl) phosphite systems (com-
pounds 9, Scheme 5; see also Table 2).

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E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
1. HC(OEt)3
HP(O)(OEt)2
PO3H2
N
N
NH2
NH2
N
N
N
H
7
PO3H2
2. HCl/H2O
H
N
1. HC(OEt)3
HP(O)(OEt)2
PO3H2
PO3H2
2. HCl/H2O
8
H
1. HC(OR)3
HP(O)(OR)2
N
PO3H
PO3H2
NH2
N
N
R
2. HCl/H2O
Fig. 2. The molecular structure and atom-numbering scheme in 9a. Displacement
ellipsoids are shown at 45% probability level.
R = Me (9a), Et (9b), Bun (9c)
Scheme 5. Reaction of trialkyl orthoformate and dialkyl phosphite with
H
N
aminopyridines.
H
N
PO3H
PO3H2
PO3H
PO3H2
N
N
H3C
H3C
Scheme 6. Mesomeric forms of compound 9a.
Table 2
Ratio of N-alkylated and non-alkylated products in the reaction of 4-aminopyridine
with various orthoformates and phosphates.
9a and indicates that this compound can exist in two mesomeric
forms (Scheme 6) with the pyridinium-type resonance form (N+–
CH3) being the major contributor to the true molecular structure.
Consistent with previous observations [14] the N1 atom of 9a is
formally sp2 hybridized and therefore both N1 and C1 atoms are
nearly coplanar with the pyridinium ring. This results in a par-
tially-double bond characters of the N1–C4 linkage, which is re-
flected in its length being 1.347(1) Å, as compared to C1–N1 of
1.463(1) Å, which is a typical value for the single C–N bond. Details
of crystal packing in 9a, determined mainly by hydrogen bonds
involving phosphonic (PO3H2) and phosphonate (PO3Hꢀ) groups,
are provided in Supplementary materials.
1. HC(OR)3
NH2
HP(O)(OR1)2
2. HCl/H2O
N
PO3H2
N
PO3H2
PO3H2
+
N
N
+
PO3H2
N
R1
R
9
9
H
N
PO3H2
PO3H2
N
11
R (R1) = Me (9a), Et (9b), Bun (9c), Prn (9d)
Entry
Substrates
HC(OR)3
Products (%)
HP(O)(OR1)
9, R
9, R1
11
Second, similarity of the 31P NMR titration curves presented in
Fig. 3 clearly indicates that the pyridinium atom N2 is alkylated
in all compounds 9a–c. This is reflected in relatively small chemical
shifts changes detected upon changing the pH, ca. 0.6 ppm upfield
in the pH range ꢁ2–5 and ca. 0.5 ppm downfield in the pH range
ꢁ5–10. Given the fact that the P(2)O3H2 group is very acidic and
releases the first proton well below pH 2, the observed changes
can be attributed to subsequent release of protons from the
1
2
3
4
5
6
7
Me
Me
Et
Et
50
100
50
0
0
20
100
50
0
0
100
100
0
0
0
50
0
0
80
0
Pri
Pri
Me
Et
Prn
Prn
Prn
Bun
Pri
Bun
100
Two possibilities of introduction of the ethyl group into bis-
phosphonate molecule can be considered, namely alkylation of
the pyridine nitrogen atom or alkylation of the amino-
methylenebisphosphonate nitrogen atom. Although the latter pos-
sibility was favored by Szabo et al. [13] our results strongly support
the former option.
First of all, the crystal structure determined for N-methyl deriv-
ative 9a unambiguously reveals the formation of product with
methylated N2 atom (Fig. 2). This compound crystallizes as a zwit-
terion with the negative charge on the phosphonate P(1)O3Hꢀ
group, the positive charge on the alkylated N2 atom and the phos-
phonic P(2)O3H2 group being neutral. It is worth to note that de-
spite similarity of the pyridinium N2–C2 and N2–C6 bond
lengths (1.358(2) and 1.356(2) Å, respectively) to those found in
the related compounds [14], the value of the endocyclic C–N–C
bond angle of 119.5(1)° is slightly below the generally accepted
limit (120°) for the endocyclic C–N–C bond angle in pyridinium
salts. This can be explained by a displacement of the charge within
16.0
15.5
15.0
compound 7
14.5
compound 8
compound 9a
14.0
compound 9b
compound 9c
13.5
13.0
12.5
12.0
11.5
0
2
4
6
8
pH
10
12
14
Fig. 3. The 31P NMR titration curves of 9a–c compared with those of 7 and 8.

E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
3811
kinetic and thermodynamic equilibrium. Thus, their ratio depends
on the specific conditions applied for an individual amine sub-
strate. The nucleophilicity seems to determine mostly the reaction
rate progress. For weak nucleophiles an excess of phosphite and
time elongation is recommended. The aforementioned factor can
effect, however, with some difficulties in the final product separa-
tion. Additional reactive groups present in the amine structure
might also be troubleshooting. Here, not typical observation con-
cerned unexpected alkylation of the pyridyl nitrogen, uniquely in
the 4-amino derivatives, was definitely evidenced and discussed.
H
N
NH2
N
C
H
HC(OEt)3
N
N
N
10
N
PO3H2
PO3H2
1. HP(O)(OEt)2
2. HCl/H2O
N
9b
Scheme 7. Two-step procedure for synthesis of 9b.
3. Experimental
P(1)O3Hꢀ and P(2)O3Hꢀ groups. This behavior is very similar to
that previously reported for piperid-1-ylmethane-1,1-diphosphon-
ic acids, characterized by the presence of the protonated piperidine
nitrogen atom over the broad range of pH [15]. Otherwise, as dem-
onstrated for 7 and 8, the proton dissociation from the pyridyl
nitrogen atom should lead to much significant changes in the 31P
NMR chemical shifts versus pH [16].
Finally, an evidence for alkylation of the pyridinium nitrogen is
provided from the 1H and 13C NMR spectra of 9a–c. A strong desh-
ielding effect exerted on the proton and carbon resonances of the
CH3 (9a) and C(10)H2 groups (9b, 9c) (see Supplementary materi-
als) may be explained by their close proximity to the positively
charged pyridinium atom N2.
In order to study the route of this reaction in some more details,
two sets of experiments were designed. First, the reaction of 4-
aminopyridine with orthoformate was found to give corresponding
intermediate N,N0-di(pyridin-4-yl)formamidine 10 with good iso-
lated yield, accordingly to the reaction mechanism described
above. The amidine 10 when reacted with diethyl phosphite fol-
lowed by acid hydrolysis afforded compound 9b in a nearly quan-
titative yield (Scheme 7). This suggested that phosphite must have
acted as an agent alkylating the pyridine nitrogen atom. Similar
alkylation of pyridines and other nitrogen containing heterocycles
by pentavalent phosphorus esters (phosphates or phosphites) were
reported previously [17]. In such substitution reactions pyridines
play a role of attacking nucleophiles whereas phosphorus monoac-
ids are appropriate leaving groups.
Second, reactions of 4-aminopyridine with various trialkyl
orthoformates and dialkyl phosphites as substrates were carried
out. Results of these studies (Table 2, relative yields of products
are given in percents as determined directly from 31P NMR spectra
of the reaction mixtures) indicated that the alkylation seems to be
a simple nucleophilic SN2 substitution [17a]. This is seen from that
only primary alkyl groups are substituting pyridine nitrogen atom
and the substitution yields follow general rules of SN2 reaction (for
example, N-methylation is favoured against N-ethylation). These
studies are somewhat complicated by the fact that phosphites re-
act with orthoformates interchanging their ester groups, and thus
providing the complex mixtures of mixed phosphite esters [18],
which in turn alkylate pyridine nitrogen.
3.1. General
All reagents and solvents were purchased from commercial
suppliers. The reaction products were identified and characterized
by their melting points with a Boetius apparatus, by analysis of
their 1H, 31P and 13C NMR, IR and MS data. NMR spectra were re-
corded on a Bruker Avance DRX-300 spectrometer operating at
300.13 MHz for 1H, 75.46 MHz for 13C and 124.50 MHz for 31P at
25 °C. Chemical shifts (d, [D6]DMSO or D2O) are reported in ppm
relative to TMS and 85% H3PO4. The standard Bruker programs
were used to perform inverse detected [1H–13C] HMQC experi-
ments that verified the 1H and 13C NMR chemical shift assign-
ments. The NMR samples for titration studies were prepared in
deuterated water. The experiments were carried out at 300 K at li-
gand concentrations 2 ꢂ 10ꢀ2 mol dmꢀ3 for 1H and 31P NMR, and
1 ꢂ 10ꢀ1 mol dmꢀ3 for 13C experiments. The pH was measured
using a Radiometer pHM 83 instrument equipped with a Mettler
Toledo INLAB 422 combined electrode and is given as readings
without correction for pD.
IR spectra were recorded on Perkin–Elmer 2000 FT spectrome-
ter in KBr pellets. Mass spectra were obtained on Bruker micrO-
TOF-Q mass spectrometer with ESI source. Reaction progress was
monitored by thin layer chromatography (TLC) on silica gel
60F254 coated aluminum. Silica gel 60 (70–230 mesh) was used
for column chromatography.
3.2. Tetraethyl (p-nitrophenylamino)methylenebisphosphonate (1):
general synthetic procedure for synthesis of methylenebisphosphonate
esters
p-Nitroaniline (30 mmol, 4.14 g), triethyl orthoformate
(30 mmol, 5.0 mL) and diethyl phosphite (60 mmol, 7.7 mL) were
refluxed for 12 h. Then volatile components were removed under
reduced pressure and the residue was worked up with acetone.
Precipitated solid was filtered and recrystallized from ethyl ace-
tate/methanol (1:5) to give 1 as yellow crystals. Yield 7.1 g, 56%;
m.p. 205 °C; dH ([D6]DMSO) 1.10 (6 H, t, J 7.0, 2 ꢂ CH3), 1.16 (6
2
H, t, J 7.0, 6H, 2 ꢂ CH3), 4.01 (8 H, m, 4 ꢂ CH2), 4.88 (1 H, dt, JHP
3
22.9, JHH 10.6, NCH), 7.05 (2 H, d, J 9.2, C6H4), 7.40 (1 H, d, J
3
10.6, HNC), 7.94 (2 H, d, J 9.2, C6H4); dC 154.01 (t, JCP 4.0, CNH),
137.42 (CNO2), 126.06 (CHCNO2), 112.81 (CHCNH), 63.3 (d, JCP
11.3, CH2), 48.44 (t, JCP 145.1, NCH), 16.70 (CH3); dP 17.53; IR
2
1
2.4. Conclusions and summary
(KBr):
m = 3268, 3088, 2982, 1596, 1506, 1477, 1314, 1287, 1235,
1113, 1022, 857 cmꢀ1; MS (ESI): m/z: 447.10 [M+Na]+, 425.1
[M+H]+; elemental Anal. Calc. for C15H26N2O8P2: C, 42.46; H,
6.18; N, 6.60; P, 14.60. Found: C, 42.39; H, 6.21; N, 6.57; P, 14.60%.
The analysis of the general mechanism of three component con-
densation of aminomethylenebisphosphonate formation was car-
ried out using p-nitroaniline as the nucleophilic substrate. The
amine was proven to be a valuable tool to follow appearance of
various intermediates, both non-phosphorus and phosphorus con-
taining. They were isolated, their structures were identified and
characterized what in turn allowed suggestion the overall reaction
route. The synthetic pathway leads to aminomethylenebisphosph-
onates via a series of imine and phosphonate products existing in
3.3. Diethyl ethoxy(p-nitrophenylamino)methanephosphonate (2)
p-Nitroaniline (30 mmol, 4.14 g), triethyl orthoformate
(30 mmol, 5.0 mL) and diethyl phosphite (60 mmol, 7.7 mL) were
refluxed gently under condenser for 1.5 h. Then volatile compo-

3812
E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
nents were removed under reduced pressure and the residue was
worked up with diethyl ether. Precipitated solid was filtered to
give 2 as yellow crystals. Yield 6.2 g, 62%; m.p. 134–135 °C; dH
([D6]DMSO) 1.18 (3 H, t, J 7.0, CH3), 1.27 (3 H, t, J 7.0, CH3), 1.35
(3 H, t, J 7.0, CH3), 3.69 (2 H, m, CH2), 4.19 (4 H, m, 2 ꢂ CH2),
Yield 5.1 g, 87%. IR, MS, elemental and melting point analyses gave
false results because of rapid decomposition of the compound.
Thus, the formamidate 5 was characterized only by NMR spectros-
copy. dH ([D6]DMSO) 1.32 (3 H, t, J 7.1, CH3), 4.28 (2 H, q, J 7.1, CH2),
7.21 (2 H, d, J 8.8, C6H4), 8.09 (1 H, s, CH), 8.19 (2 H, d, J 8.8, C6H4).
2
3
5.06 (1 H, dd, JHP JHH 10.0, NCH), 5.60 (1 H, m, HNC), 6.76 (2 H,
3
d, J 9.1, C6H4), 8.09 (2 H, d, J 9.1, C6H4); dC 153.61 (d, JCP 10.1,
3.7. General procedure for synthesis of methylenebisphosphonic acids,
aminopyridine derivatives [10c]
CNH), 138.29 (CNO2), 126.19 (CHCNO2), 113.19 (CHCNH), 79.03
1
2
(d, JCP 198.4, NCH), 63.90 (d, JCP 11.4, CH2), 63.11 and 63.03 (d
2
2
3
each, JCP 11.8 and JCP 11.7, CH2), 16.80 (d, JCP 4.3, CH3), 15.49
Round-bottom flask (50 mL) was charged with aminopyridine
(30 mmol, 2.82 g), triethyl orthoformate (30 mmol, 5.0 mL) and
diethyl phosphite (60 mmol, 7.7 mL). The mixture was refluxed
for 12 h. Volatile components of the reaction mixture were re-
moved under reduced pressure and the oily residue was dissolved
in 20 mL of concentrated hydrochloric acid and refluxed for 8 h.
Then the solvent was removed in vacuo and the obtained product
was purified by crystallization from water/ethanol mixture.
(CH3); dP 16.88; IR (KBr):
m = 3262, 3205, 3078, 2978, 1598, 1550,
1505, 1489, 1315, 1229, 1113, 1066, 1022, 844 cmꢀ1; MS (ESI):
m/z: 355.10 [M+Na]+, 333.10 [M+H]+; elemental Anal. Calc. for
C13H21N2O6P: C, 46.99; H, 6.37; N, 8.43; P, 9.32. Found: C, 46.52;
H, 6.17; N, 8.67; P, 9.36%.
Diethyl ether solution obtained after filtration still contained
not separated 2 and all the remaining intermediates (compounds
3–5, Rf = 0.13 for 3, Rf = 0.33 for 5, Rf = 0.63 for 4, TLC in ethyl ace-
tate/hexane, 3:1). After removal of the solvent the residue was sub-
jected to column chromatography in ethyl acetate/hexane gradient
(1:3 to 1:0) to yield the analytical samples. For conditions of the
preparative syntheses see below.
3.8. N-(1-Methylpyridinium-4-amino)methylenebisphosphonic acid
(9a)
Yield 2.9 g, 43% (from reaction presented as an entry 2 in Table
2); m.p. 305–307 °C; dH (D2O, pD = 6.64) 3.80 (3 H, s, CH3), 3.89 (1
2
3.4. Diethyl bis(p-nitrophenylamino)methanephosphonate (3)
H, t, JPH 19.3, NCH), 6.84, 7.74, 7.89 (2 H, 1 H and 1 H, m each,
3
4 ꢂ CHpy); dC 156.71 (d, JPC 4.7, C4py), 143.98, 141.66 (C2py and
1
N,N0-Di(p-nitrophenyl)formamidine (4) (30 mmol, 8.6 g) and
diethyl phosphite (90 mmol, 11.5 mL) were heated at 90 °C (an
oil bath) under condenser for 6 h. Then volatile components were
removed under reduced pressure and the residue was treated with
acetone. Precipitated solid was filtered and recrystallized from
dioxane/toluene (2:1) to give 3 as yellow crystals. Yield 9.4 g,
74%; m.p. 176 °C; dH ([D6]DMSO) 1.16 (6 H, t, J 7.1, 2 ꢂ CH3),
C6py), 111.33, 105.99 (C3py and C5py), 52.06 (t, JPC 123.8, NCH),
44.36 (CH3); dP 13.53; MS (ESI): m/z: 281.8 [MꢀH]+; elemental
Anal. Calc. for C7H12N2O6P2: C, 29.77; H, 4.25; N, 10.06; P, 22.89.
Found: C, 29.35; H, 4.42; N, 10.06; P, 21.89%.
3.9. N-(1-Ethylpyridinium-4-amino)methylenebisphosphonic acid
(9b)
2
3
4.06 (4 H, m, 2 ꢂ CH2), 5.66 (1 H, dt, JHP 16.4, JHH 8.3, NCH),
3
3
6.94 (4 H, d, J 9.3, 2 ꢂ C6H4), 7.69 (2 H, dd, JHP 8.3, JHH 4.6,
Yield 5.4 g, 61% (from reaction presented in Scheme 5) and 81%
(from reaction presented in Scheme 7); m.p. 287–289 °C; dH (D2O,
pD = 4.88) 1.37 (3 H, t, J 7.2, CH3), 4.02 (1 H, t, 2JPH 19.8, NCH), 4.08
(2 H, q, J 7.2, CH2), 6.86, 6.90, 7.86, 8.01 ppm (1 H each, m each,
3
2 ꢂ NHC), 7.99 (4 H, d, J 9.3, 2 ꢂ C6H4); dC 153.08 (d, JCP 8.6,
CNH), 137.99 (CNO2), 126.22 (CHCNO2), 113.02 (CHCNH), 63.50
2
1
3
(d, JCP 6.9, CH2), 58.83 (d, JCP 181.65, NCH), 16.79 (d, JCP 5.1,
CH3); dP 16.08; IR (KBr): = 3348, 3265, 3063, 2987, 1604, 1505,
3
m
4 ꢂ CHpy); dC 156.94 (d, JPC 4.4, C4py), 142.94, 140.68 (C2py and
1
1479, 1471, 1526, 1472, 1320, 1301, 1265, 1107, 1048, 1020,
838 cmꢀ1; MS (ESI): m/z: 447.10 [M+Na]+, 425.1 [M+H]+; elemental
Anal. Calc. for C17H21N4O7P: C, 48.12; H, 4.99; N, 13.20; P, 7.30.
Found: C, 47.77; H, 4.78; N, 13.04; P, 7.30%.
C6py), 111.61, 106.19 (C3py and C5py), 53.28 (CH2), 51.64 (t, JPC
129.5, NCH), 15.10 (CH3); dP 13.18; MS (ESI): m/z: 297.1 [M]+; ele-
mental Anal. Calc. for C8H14N2O6P2: C, 32.42; H, 4.73; N, 9.46; P,
20.94. Found: C, 31.84; H, 4.79; N, 8.98; P, 21.08%.
3.5. N,N0-Di(p-nitrophenyl)formamidine (4)
3.10. N-(1-n-Butylpyridinium-4-amino)methylenebisphosphonic acid
(9c)
p-Nitroaniline (30 mmol, 4.14 g) and triethyl orthoformate
(15 mmol, 2.49 mL) were heated at 90 °C (an oil bath) in o-xylene
(10 mL) under condenser for 8 h. Then the solvent and other vola-
tile components were removed under reduced pressure. The resi-
due was worked up with toluene and precipitated solid was
filtered to give 4 as yellow crystals. Yield 6.7 g, 78%; m.p. 241 °C;
dH ([D6]DMSO) 6.68 (4 H, br d, 2 ꢂ C6H4), 8.11 (4 H, d, J 9.1,
2 ꢂ C6H4), 8.49 (1 H, br s, HNC), 10.75 (1 H, br s, CH); dC 153.75
(C@N), 150.48 (CNH), 143.40 (CNO2), 125.61 (CHCNO2), 122.30
Yield 5.2 g, 54% (from reaction presented as an entry 7 in Table
2); m.p. 305–307 °C; dH (D2O, pD = 5.52) 0.87 (3 H, t, J 7.2, CH3),
2
1.27 (2 H, m, CH2), 1.78 (2 H, m, CH2), 3.98 (1 H, t, JPH 18.3,
NCH), 4.08 (2 H, t, J 7.2, CH2), 6.91, 6.92, 7.88, 8.02 (1 H each, m
3
each, 4 ꢂ CHpy); dC 156.40 (d, JPC 2.1, C4py), 143.31, 140.47 (C2py
and C6py), 111.58, 106.07 (C3py and C5py), 57.89 (CH2), 51.78 (t,
1JPC 124.5, NCH), 32.04 (CH2), 18.72 (CH2), 12.75 (CH3); dP 12.48;
MS (ESI): m/z: 325.0 [M]+; elemental Anal. Calc. for C10H18N2O6P2:
C, 37.02; H, 5.55; N, 8.64; P, 19.013. Found: C, 36.73; H, 5.27; N,
8.86; P, 18.70%.
(CHCNH), 117.58 (CHCNH); IR (KBr):
m = 3304, 3179, 3104, 3036,
1660, 1599, 1579, 1522, 1508, 1344, 1312, 1300, 1205, 846 cmꢀ1
;
MS (ESI): m/z: 287.10 [M+H]+; elemental Anal. Calc. for
C13H10N4O4: C, 54.55; H, 3.52; N, 19.57. Found: C, 54.50; H, 3.54;
N, 19.54%.
3.11. N,N0-Di(pyridin-4-yl)formamidine (10)
4-Aminopyridine (30 mmol, 2.82 g) and of triethyl orthofor-
mate (20 mL) were refluxed under condenser for 12 h. Then vola-
tile components were removed under reduced pressure and
resulting yellow oil dissolved in water. Compound 10 of satisfac-
tory purity crystallized as yellowish solid. Yield 4.9 g 82%, m.p.
105–107 °C; dH (D2O) 6.21 (4 H, d, J 7.1, Py), 7.34 (4H, d, J 7.1,
Py), 7.61 (1 H, s, N@CH–N); MS (ESI): m/z: 199.4 [M+H]+; elemental
3.6. Ethyl N-(p-nitrophenyl)formimidate (5)
p-Nitroaniline (30 mmol, 4.14 g) and triethyl orthoformate
(150 mmol, 25.0 mL) were heated at 90 °C (an oil bath) under con-
denser for 0.5 h. The residue was cooled and worked up with ace-
tone. Precipitated solid was filtered to give 5 as yellow crystals.

E. Da˛browska et al. / Journal of Organometallic Chemistry 694 (2009) 3806–3813
3813
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Acknowledgment
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Financial support by Ministry of Science and Higher Education
(Grant No. R05 034 03) is gratefully acknowledged.
(b) N. Rodriguez, B.N. Bailey, M.B. Martin, E. Oldfield, J.A. Urbina, R. Docampo,
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Appendix A. Supplementary material
(c) M.B. Martin, J.M. Sanders, H. Kendrick, K. de Luca-Fradley, J.C. Lewis, J.M.
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