Three-Component Reaction of Diamines with Triethyl Orthoformate and Diethyl Phosphite and Anti-Proliferative and Antiosteoporotic Activities of the Products

A three-component reaction between diamines (diaminobenzenes, diaminocyclohexanes, and piperazines), triethyl orthoformate, and diethyl phosphite was studied in some detail. In the case of 1,3- and 1,4-diamines and piperazines, products of the substitution of two amino moieties—the corresponding tetraphosphonic acids—were obtained. In the cases of 1,2-diaminobenzene, 1,2-diaminocyclohexanes and 1,2-diaminocyclohexenes, only one amino group reacted. This is most likely the result of the formation of hydrogen bonding between the phosphonate oxygen and a hydrogen of the adjacent amino group, which caused a decrease in the reactivity of the amino group. Most of the obtained compounds inhibited the proliferation of RAW 264.7 macrophages, PC-3 human prostate cancer cells, and MCF-7 human breast cancer cells, with 1, trans-7, and 16 showing broad nonspecific activity, which makes these compounds especially interesting in the context of anti-osteolytic treatment and the blocking of interactions and mutual activation of osteoclasts and tumor metastatic cells. These compounds exhibit similar activity to zoledronic acid and higher activity than incadronic acid, which were used as controls. However, studies of sheep with induced osteoporosis carried out with compound trans-7 did not support this assumption.


Introduction
Although the synthesis of bisphosphonic acids was described as early as the 19th century, the interest in these compounds is still growing, rooted in their promising and variable physiological activities [1]. Most commonly, their regulation of bone growth has been exploited for the design and preparation of antiosteoporotic drugs that can also be used to treat skeletal complications of Molecules 2020, 25 various provenances [1][2][3][4]. These compounds also exert an inhibitory activity towards important medicinal enzymes, including farnesyl pyrophosphate synthase (thus inhibiting Ras farnesylation, as their structure makes them potential anti-cancer and/or anti-protozoan agents), glutamine synthetase (possible anti-tuberculosis agents), and undecaprenyl diphosphate synthase (antibacterials) [1,[5][6][7][8][9]. Some patients who received intravenous and oral forms of bisphosphonate therapy for various bone-related conditions suffer osteonecrosis of the jaw, dependent on the dose and duration of the therapy [10]. The mechanism by which bisphosphonates might contribute to the development of osteonecrosis of the jaw is not well understood [11]. However, it may result, at least in part, from the high binding and long residence of bisphosphonates in the bone tissue [12], with two phosphonate groups and an adjacent hydroxyl moiety mediating the binding [13]. Thus, it has been suggested that their analogues with a lower affinity for bone minerals could be useful. The use of aminomethylenebisphosphonic acids lacking the hydroxyl moiety seems to be obvious solution to reduce the bone affinity and increase the bioavailability of bisphosphonates. We have synthesized hundreds of these compounds and screened their anti-proliferative activity towards mouse macrophage J774E cells [14]. Since these cells originate from the same precursors as osteoclasts, this test is considered a reasonable preliminary test for antiosteoporotic activity.
In this paper, we describe the application of a commonly used a three-component reaction of amines with triethyl orthoformate and diethyl phosphite [15][16][17] We present the reactions with diamines (diaminobenzenes, diaminocyclohexanes, diaminocyclohexenes and piperazines) being used as substrates. Since this reaction usually gives mixtures of products that are difficult to separate, the crude esters are commonly hydrolyzed with concentrated hydrochloric acid and the desired bisphosphonic acids were isolated in moderate to good yields. The anti-proliferative action of these compounds against mouse macrophage-like RAW 264.7 cells, originating from the same precursors as osteoclasts, was screened. As a result, trans-cyclohexane-1,4-di(aminomethylenebisphosphonic) acid (trans-7) was chosen for the study of its antiosteoporotic activity on sheep. Additionally, the anti-cancer potency of the synthesized compounds was studied using the MCF-7 cell line, which is a well-established model of breast cancer, and the PC-3 human prostate cancer cell line.

Reactions of Diaminobenzenes
The reaction of 1,4-diaminobenzene with triethyl orthoformate and diethyl phosphite ( Figure 1) readily provided 1,4-di-substituted tetrabisphosphonic acid (compound 1), which appeared to be relatively stable if stored at low temperature while protected against moisture. When 1,2-diaminobenzene was used, a mixture of products containing mono-and di-substituted compounds (compounds 2 and 3, respectively) was obtained, with the mono-substituted produce the major one (as revealed from NMR and MS studies- Figure 2 and Supplementary Data), although we did not succeed in their isolation from the reaction mixture. 1,3-Diaminobenzene gave a complex mixture of inseparable products, most likely di-(predominating) and mono-substituted compounds (compounds 4 and 5) accompanied by several side products. The formation of structurally variable side products is not surprising since it was frequently observed earlier in the three-component condensation of amines with trialkyl orthoformates and diethyl phosphites [15][16][17][18]. The efforts undertaken to separate and isolate the products of the reactions of 1,2-and 1,3-diaminobenzenes failed since the postreaction mixtures turned dark violet over time, and NMR studies revealed the intense decomposition of their components.

Reactions of Diaminocyclohexanes
Diaminocyclohexanes are valuable intermediates to produce dyestuffs, textile assistants, fungicides, pesticides, and pharmaceuticals and thus are commercially available in all stereoisomeric forms. We decided to undertake a more detailed study of the reactions in which the stereoisomers of diaminocyclohexanes were used as substrates. As expected, in the cases of cyclohexane-1,3-diamine and cyclohexane-1,4-diamine, two amino groups reacted ( Figure 3). Thus, the reaction of a racemic mixture of cis,trans-cyclohexane-1,3-diamine provided compound 6, seen as a doublet in the 31 P NMR spectrum. This doublet is derived from the presence of a diastereomeric mixture composed of a meso isomer obtained from the cis-substrate and a mixture of enantiomers formed from the trans-substrate. The reaction of (±)-trans-cyclohexane-1,4-diamine gave meso-compound trans-7, as seen by the presence of a singlet in the 31 P NMR spectrum.

Reactions of Diaminocyclohexanes
Diaminocyclohexanes are valuable intermediates to produce dyestuffs, textile assistants, fungicides, pesticides, and pharmaceuticals and thus are commercially available in all stereoisomeric forms. We decided to undertake a more detailed study of the reactions in which the stereoisomers of diaminocyclohexanes were used as substrates. As expected, in the cases of cyclohexane-1,3-diamine and cyclohexane-1,4-diamine, two amino groups reacted ( Figure 3). Thus, the reaction of a racemic mixture of cis,trans-cyclohexane-1,3-diamine provided compound 6, seen as a doublet in the 31 P NMR spectrum. This doublet is derived from the presence of a diastereomeric mixture composed of a meso isomer obtained from the cis-substrate and a mixture of enantiomers formed from the trans-substrate. The reaction of (±)-trans-cyclohexane-1,4-diamine gave meso-compound trans-7, as seen by the presence of a singlet in the 31 P NMR spectrum.   In the case of cyclohexane-1,2-diamines, only one amino group reacted, yielding compound 8, independent of the ratio of substrates being used. By using all the available stereoisomers of diaminocyclohexane, as well as their mixtures, we have proven that the absolute configuration is retained ( Figure 4). Quite interestingly the differences in the 1 H and 13 C NMR spectra were small if not negligible (see Supplementary Data). In the 31 P NMR, a slightly broadened doublet was observed for enantiomerically pure products-(1S,2S)-and (1R,2R)-cyclohexane-1-amino-2-aminomethylenebisphosphonic acids. This suggests that the phosphonic groups are magnetically nonequivalent. We speculate that this may result either from the conformation of compound 8 being frozen by the formation of intramolecular hydrogen bonding between the amino proton and phosphonic acid oxygen or from formation of strong hydrogen bonded dimers of this bisphosphonate. An identical result was obtained when using (1S,2S)-(+)-cyclopentane-1,2-diamine as a substrate ( Figure 4).   Figure 3. Products of the reactions of cyclohexane-1,3-and 1,4-diamines with triethyl orthoformate and diethyl phosphite.
In the case of cyclohexane-1,2-diamines, only one amino group reacted, yielding compound 8, independent of the ratio of substrates being used. By using all the available stereoisomers of diaminocyclohexane, as well as their mixtures, we have proven that the absolute configuration is retained ( Figure 4). Quite interestingly the differences in the 1 H and 13 C NMR spectra were small if not negligible (see Supplementary Data). In the 31 P NMR, a slightly broadened doublet was observed for enantiomerically pure products-(1S,2S)and (1R,2R)-cyclohexane-1-amino-2-aminomethylenebisphosphonic acids. This suggests that the phosphonic groups are magnetically nonequivalent. We speculate that this may result either from the conformation of compound 8 being frozen by the formation of intramolecular hydrogen bonding between the amino proton and phosphonic acid oxygen or from formation of strong hydrogen bonded dimers of this bisphosphonate. An identical result was obtained when using (1S,2S)-(+)-cyclopentane-1,2-diamine as a substrate ( Figure 4).
The use of (±)-trans-1,2-diaminocyclohexane and an equimolar mixture of the cis and trans isomers as substrates resulted, as expected, in more complex 31 P NMR spectra ( Figure 5 and Supplementary Data), which reflects the growing number of stereoisomers. The use of (±)-trans-1,2-diaminocyclohexane and an equimolar mixture of the cis and trans isomers as substrates resulted, as expected, in more complex 31 P NMR spectra ( Figure 5 and Supplementary Data), which reflects the growing number of stereoisomers. 17      The use of (±)-trans-1,2-diaminocyclohexane and an equimolar mixture of the cis and trans isomers as substrates resulted, as expected, in more complex 31 P NMR spectra ( Figure 5 and Supplementary Data), which reflects the growing number of stereoisomers. 17    The magnetic nonequivalence of the phosphonic groups is also pronounced when (1S,2S)-and (1R,2R)-4-cyclohexene-1,2-diamines and (±)-trans-4-cyclohexene-1,2-diamine were used as substrates ( Figure 6). In this case, the 31 P NMR spectrum of (±)-trans-4-cyclohexene-1-amino-2-amino-methylenebisphosphonic acid (compound trans-10) reveals two clearly separated doublets ( Figure 6), whereas for the enantiomeric forms, broadened doublets are seen.

Reactions of Piperazines
The secondary amino groups of piperazine reacted smoothly, yielding piperaz-1,4-diylmethylenebisphosphonic acid 11. The mono-substituted derivative 12 was obtained by using N-acetylpiperazine as a substrate (Figure 7). Stereoisomers of 2,5-dimethylpiperazine and 2,6-dimethylpiperazine were also used to provide bisphosphonates 13 and 14, respectively ( Figure  7). While all four phosphorus atoms in compound 11 are magnetically equivalent, which is demonstrated by the singlet in the 31 P NMR spectrum, the spectra of compounds 13 and 14 are more complex. This is because the introduction of steric hindrance in proximity to the aminomethylenebisphosphonic substituent results in the nonequivalence of the phosphonic moieties, which is seen by the appearance of an AB spin system. Quite interestingly, the spectrum of compound cis-14 is composed of both an AB system, derived from the substitution at position 1, and  While all four phosphorus atoms in compound 11 are magnetically equivalent, which is demonstrated by the singlet in the 31 P NMR spectrum, the spectra of compounds 13 and 14 are more complex. This is because the introduction of steric hindrance in proximity to the aminomethylenebisphosphonic substituent results in the nonequivalence of the phosphonic moieties, which is seen by the appearance of an AB spin system. Quite interestingly, the spectrum of compound cis-14 is composed of both an AB system, derived from the substitution at position 1, and a singlet, resulting from the substitution at position 4 ( Figure 8). In the spectrum of rac-14, only the substituents at position 4 differentiate the isomers to enable the determination that a non-equimolar mixture of the cis and trans isomers was obtained.
Molecules 2020, 25, x 8 of 19 a singlet, resulting from the substitution at position 4 ( Figure 8). In the spectrum of rac-14, only the substituents at position 4 differentiate the isomers to enable the determination that a non-equimolar mixture of the cis and trans isomers was obtained.

Reactions of Aminoalkylidenediamines
To enlarge the set of compounds chosen for biological studies, it was additionally supplemented by compounds 15 and 16 ( Figure 9). However, we were not able to obtain ethylene-di(aminomethylenebisphosphonic acid) 15 in a pure form since this reaction is accompanied by the ethylation of one amino group to yield compound 17. Compounds 15 and 17 are difficult to separate, so we decided to perform the biological studies using the impure derivative.

In Vitro Evaluation
To screen for the potential antiosteoporotic activity of the bisphosphonates, their anti-proliferative activity towards in vitro cell cultures was determined. Because of the difficulty in isolating and culturing large numbers of osteoclasts, most studies devoted to the characterization of the pharmacological properties of bisphosphonates in vitro are performed on osteoclast surrogates, particularly macrophages [19,20]. Both RAW 264.7 macrophages and osteoclasts are derived from a hematopoietic lineage and are highly endocytic and capable of demineralizing bone particles [21,22]. They are well recognized for being sensitive to bisphosphonates, which most likely act by inducing apoptosis. The results summarized in Table 1 clearly show that nearly all the studied compounds effectively inhibit the proliferation of RAW 264.7 macrophages, exhibiting potency similar or slightly higher than incadronate and zoledronate, which are popular antiosteoporotic drugs. The only exceptions are compounds (1S,2S)-9 and rac-14, which are an order of magnitude less active. The most active appeared to be compounds 1, rac-6, and 11.

Reactions of Aminoalkylidenediamines
To enlarge the set of compounds chosen for biological studies, it was additionally supplemented by compounds 15 and 16 ( Figure 9). However, we were not able to obtain ethylene-di(aminomethylenebisphosphonic acid) 15 in a pure form since this reaction is accompanied by the ethylation of one amino group to yield compound 17. Compounds 15 and 17 are difficult to separate, so we decided to perform the biological studies using the impure derivative. a singlet, resulting from the substitution at position 4 ( Figure 8). In the spectrum of rac-14, only the substituents at position 4 differentiate the isomers to enable the determination that a non-equimolar mixture of the cis and trans isomers was obtained.

Reactions of Aminoalkylidenediamines
To enlarge the set of compounds chosen for biological studies, it was additionally supplemented by compounds 15 and 16 ( Figure 9). However, we were not able to obtain ethylene-di(aminomethylenebisphosphonic acid) 15 in a pure form since this reaction is accompanied by the ethylation of one amino group to yield compound 17. Compounds 15 and 17 are difficult to separate, so we decided to perform the biological studies using the impure derivative.

In Vitro Evaluation
To screen for the potential antiosteoporotic activity of the bisphosphonates, their anti-proliferative activity towards in vitro cell cultures was determined. Because of the difficulty in isolating and culturing large numbers of osteoclasts, most studies devoted to the characterization of the pharmacological properties of bisphosphonates in vitro are performed on osteoclast surrogates, particularly macrophages [19,20]. Both RAW 264.7 macrophages and osteoclasts are derived from a hematopoietic lineage and are highly endocytic and capable of demineralizing bone particles [21,22]. They are well recognized for being sensitive to bisphosphonates, which most likely act by inducing apoptosis. The results summarized in Table 1 clearly show that nearly all the studied compounds effectively inhibit the proliferation of RAW 264.7 macrophages, exhibiting potency similar or slightly higher than incadronate and zoledronate, which are popular antiosteoporotic drugs. The only exceptions are compounds (1S,2S)-9 and rac-14, which are an order of magnitude less active. The most active appeared to be compounds 1, rac-6, and 11.

In Vitro Evaluation
To screen for the potential antiosteoporotic activity of the bisphosphonates, their anti-proliferative activity towards in vitro cell cultures was determined. Because of the difficulty in isolating and culturing large numbers of osteoclasts, most studies devoted to the characterization of the pharmacological properties of bisphosphonates in vitro are performed on osteoclast surrogates, particularly macrophages [19,20]. Both RAW 264.7 macrophages and osteoclasts are derived from a hematopoietic lineage and are highly endocytic and capable of demineralizing bone particles [21,22]. They are well recognized for being sensitive to bisphosphonates, which most likely act by inducing apoptosis. The results summarized in Table 1 clearly show that nearly all the studied compounds effectively inhibit the proliferation of RAW 264.7 macrophages, exhibiting potency similar or slightly higher than incadronate and zoledronate, which are popular antiosteoporotic drugs. The only exceptions are compounds (1S,2S)-9 and rac-14, which are an order of magnitude less active. The most active appeared to be compounds 1, rac-6, and 11. Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, 215. 8  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, Molecules 2020, 25, x 10 of 19 Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, Molecules 2020, 25, x 10 of 19 Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, although with IC50 values that are significantly higher (including those of the controls). These studies also indicated that compound 1 was the most promising candidate for further studies.
Taking into account the possible clinical application compounds, 1, trans-7, and 16 are the most interesting of all the compounds tested. They show broad nonspecific activity, higher than that  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work,  Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, Since bisphosphonates accumulate in the bones, a strong anti-proliferative effect on bone metastatic and hematopoietic tumors can be expected, suggesting their possible clinical application as anti-cancer drugs. Therefore, we also tested the action of the synthesized compounds on the two classic cancer cell lines MCF-7 and PC-3. MCF-7 is a well-established model of breast cancer, which preferentially metastasizes to the bone, forming predominantly osteolytic lesions, whereas PC-3 is a human prostate cancer cell line used in prostate cancer research and drug development.
All aliphatic bis-and tetraphosphonates showed poor anti-proliferative activity against the prostate cell line PC-3, except for compound rac-14. Quite interestingly compound rac-14 was as potent as cisplatin while being only weakly active against the remaining cell lines, thus showing marked selectivity.
However, the aromatic bis[aminomethylene(bisphosphonic)] acid (1) showed a similar or higher activity than the reference bisphosphonates for the PC-3 line. Compounds 1, 6 and 11 were the most active (Table 1) against the macrophage cell line RAW 264.7. Compound 1 showed a broad anti-proliferative activity towards all cells applied in the study. It inhibited the proliferation of MCF-7, RAW 264.7 and PC-3 cells with IC 50 values ranging from approximately 5 µM (RAW 264.7) to 216 µM (PC-3). The IC 50 value for PC-3 was one and half-fold lower than the corresponding value for zoledronic acid and in the same range as that for incadronic acid but worse than that of cisplatin. For the RAW 264.7 macrophages, compound 1 showed a nine times better anti-proliferative activity than zoledronate and a ten times higher activity than incadronate.
Similar studies of the anti-proliferative activity of this class of compounds towards the J774E, MC-7 and HL60 cell lines have been recently published [23]. The described set included compounds 1, trans-7, and 16. The results of these studies are more or less similar to those presented in this work, although with IC 50 values that are significantly higher (including those of the controls). These studies also indicated that compound 1 was the most promising candidate for further studies.
Taking into account the possible clinical application compounds, 1, trans-7, and 16 are the most interesting of all the compounds tested. They show broad nonspecific activity, higher than that found for incadronic acid, influencing the proliferation of RAW 264.7 macrophages and the PC-3 and MCF-7 cell lines. Compound 1 shows a broad nonspecific activity that is higher than that of incadronic acid. Compound 1 specifically and strongly influences the proliferation of RAW 264.7 macrophages and the PC-3 and MCF-7 lines. This is especially interesting in the context of anti-osteolytic treatment and the blocking of the interactions and mutual activation of osteoclasts and tumor metastatic cells in the bone microenvironment.

In vivo Evaluation of Compounds
Compounds 1, trans-7, and 16 were the logical options for the treatment of sheep with induced osteoporosis. These animals were chosen due to the similarity of sheep to humans in weight, bone and joint structure, and bone regeneration mechanisms. Based on the previous literature report and our studies, compound 1 seemed to be the best choice. However, it is unstable in the solution prepared for application in sheep [24,25]. This is clearly visible from the solution turning violet, and 31 P NMR spectra taken versus time indicate the decomposition of the carbon-to phosphorus bond to form phosphorous acid. Therefore, compound trans-7 was chosen for in vivo studies, taking into consideration its stability and solubility in aqueous solutions. Because osteoporosis does not occur naturally in animals, it must be induced in sheep. It was induced by a separately described regimen based on ovariectomy followed by controlled methylprednisolone treatment with respect to bone metabolism and a suitable calcium/vitamin D-restricted diet [26]. The level of bone loss was determined by measuring the bone mineral density by means of quantitative computed tomography (QCT) and computer radiography at the PET-TK Laboratory, Medical Diagnostic Center in Kraków. The bone structural parameters were determined from iliac crest biopsy specimens using a diamond trephine.
After two months, the administration of bisphosphonate was started. An aqueous solution of the sodium salt of bisphosphonate trans-7 (35 mg/sheep) was given to the animals every week using a probe directly inserted into the rumen. Ten doses of the drug were given to five out of seven sheep. One sheep was not medicated (negative control), whereas one was healthy (no osteoporosis was induced here-positive control). At the end, the animals were sacrificed, and bone samples were collected from the shaft, total hip, and radius of the femur and the proximal and distal tibial epiphysis for histology. This examination revealed the presence of small erosion bays, where no osteogenesis was observed, and large osteons characterized by arrested osteogenesis (Figure 10 and Supplementary Data). In some bone lamella, a dysfunctional course of collagen fibers was noted, which indicates an inability to finish the bone turnover process.

In vivo Evaluation of Compounds
Compounds 1, trans-7, and 16 were the logical options for the treatment of sheep with induced osteoporosis. These animals were chosen due to the similarity of sheep to humans in weight, bone and joint structure, and bone regeneration mechanisms. Based on the previous literature report and our studies, compound 1 seemed to be the best choice. However, it is unstable in the solution prepared for application in sheep [24,25]. This is clearly visible from the solution turning violet, and 31 P NMR spectra taken versus time indicate the decomposition of the carbon-to phosphorus bond to form phosphorous acid. Therefore, compound trans-7 was chosen for in vivo studies, taking into consideration its stability and solubility in aqueous solutions. Because osteoporosis does not occur naturally in animals, it must be induced in sheep. It was induced by a separately described regimen based on ovariectomy followed by controlled methylprednisolone treatment with respect to bone metabolism and a suitable calcium/vitamin D-restricted diet [26]. The level of bone loss was determined by measuring the bone mineral density by means of quantitative computed tomography (QCT) and computer radiography at the PET-TK Laboratory, Medical Diagnostic Center in Kraków. The bone structural parameters were determined from iliac crest biopsy specimens using a diamond trephine.
After two months, the administration of bisphosphonate was started. An aqueous solution of the sodium salt of bisphosphonate trans-7 (35 mg/sheep) was given to the animals every week using a probe directly inserted into the rumen. Ten doses of the drug were given to five out of seven sheep. One sheep was not medicated (negative control), whereas one was healthy (no osteoporosis was induced here-positive control). At the end, the animals were sacrificed, and bone samples were collected from the shaft, total hip, and radius of the femur and the proximal and distal tibial epiphysis for histology. This examination revealed the presence of small erosion bays, where no osteogenesis was observed, and large osteons characterized by arrested osteogenesis (Figure 10 and Supplementary Data). In some bone lamella, a dysfunctional course of collagen fibers was noted, which indicates an inability to finish the bone turnover process.
Summing up, a mild antiosteoporotic activity of compound trans-7, compared to incadronate (results not shown), has been found, and thus these results are considered unsatisfactory. Summing up, a mild antiosteoporotic activity of compound trans-7, compared to incadronate (results not shown), has been found, and thus these results are considered unsatisfactory.

General Information
All solvents and reagents were purchased from commercial suppliers, were of analytical grade and were used without further purification. Unless otherwise specified, solvents were removed with a rotary evaporator. The 1 H-, 31 P-and 13 13 C). Measurements were made in solutions of D 2 O + NaOD at 300 K, and all solvents were supplied by ARMAR AG (Dottingen, Switzerland). Chemical shifts are reported in ppm relative to TMS (tetramethylsilane) and 85% H 3 PO 4 , used as external standards, and coupling constants are reported in Hz. Melting points were determined on an SRS Melting Point Apparatus OptiMelt MPA 100 (Stanford Research Systems, Sunnyvale, CA, USA) and are reported uncorrected. The purity of all test compounds is higher than 95% by 1 H NMR and LC-MS. Mass spectra were recorded at the Faculty of Chemistry, Wrocław University of Science and Technology using a Waters LCT Premier XE mass spectrometer (method of electrospray ionization, ESI) (Waters, Milford, MA, USA).

Synthetic Procedures
3.2.1. General Procedure for the Synthesis of Aliphatic Bisaminomethylenebisphosphonates (6,(8)(9)(10)(11)16): Method A A mixture of an aliphatic diamine (0.03 mol) and the appropriate amounts of diethyl phosphite (0.126 mol, 16.32 mL) and triethyl orthoformate (0.063 mol, 4.4 mL) was heated with stirring at an elevated temperature (130 • C) for 15 h (overnight) on the heating plate of a Radleys Carousel. Then, the volatile components of the reaction mixture were evaporated, and the residue was dissolved in chloroform (200 mL). The organic layer was extracted three times with water (3 × 150 mL), and the inorganic layer was evaporated under reduced pressure. The crude esters were then subjected to hydrolysis. (1-7,trans-8, 11-14): Method B Diamine (0.03 mol) and the appropriate amounts of diethyl phosphite (0.126 mol, 16.32 mL) and triethyl orthoformate (0.063 mol, 4.4 mL) were heated and simultaneously stirred at a temperature of 130 • C on a heating plate (125 • C in the reaction medium) of a Radleys Carousel apparatus overnight (15 h). The mixture was cooled, and the volatile components were removed using a rotary evaporator. The resulting mixture was dissolved in ethyl acetate (100 mL) and purified by washing with water (100 mL), saturated sodium chloride solution (100 mL) and again with water (100 mL). The solution was dried over anhyd MgSO4, and the solvent was evaporated under vacuum. The crude esters were then subjected to hydrolysis.

Hydrolysis-General Procedure
The obtained ester (0.030 mol) was refluxed for 15 h (overnight) in 40 mL 6 M aqueous hydrochloric acid solution. After cooling, the volatile components were removed using a rotary evaporator, and the resulting oil was dissolved in a minimal amount of water (40-50 mL), decolored with activated charcoal and purified by crystallization from a water-ethanol mixture (80/20 v/v). The impure product was mixed with water for a few days until the dissolution of impurities was observed and then was filtered and washed with distilled water and dried in vacuo.
trans-Cyclohexane-1,4-di(aminomethylenebisphosphonic) acid (trans-7) [14,23,27] was obtained as a white solid; yield: 52% (method B); mp 243 • C. All studies were conducted in accordance with the Wrocław 2nd Local Ethics Committee for Animal Experiments (resolution no 85/2009 of 27.02.2009) and was reviewed at and the study protocol was approved by this ethics committee.

Histological Studies
The bone samples underwent conservation in 4% formalin solution and were decalcified by successive bathing in 10% sodium versenate, distilled water, a mixture of formic and citric acids, and distilled water. Then, they were dehydrated and kept in paraffin. The bone segments were sectioned with a microtome to 6-7 µm slides for histomorphometric analysis. Before the analysis, the samples were fixed for 3 h in a 3.5% solution of glutaraldehyde in phosphate buffer (0.1 M, pH between 7.2 and 7.4), washed thoroughly with buffer and then fixed with a solution containing 1.0% osmium tetroxide in the same buffer. After drying (ethanol-acetone), the samples were submerged in Epon 812.
The bone slides were stained by the Delafield method with hematoxylin and eosin and analyzed by transmission microscopy (Tesla BS-500).

Conclusions
A three-component reaction between 1,4-diamines, triethyl orthoformate, and diethyl phosphite resulted in introduction of two aminomethylenebisphosphonate moieties into the molecule. When 1,2-diamines were used as the substrates, the reaction surprisingly provided mono-substituted aminomethylenebisphosphonates despite the ratio of the substrates used most likely, as a result of steric hindrance. Moreover, stability of the mono-substituted products is enhanced by the formation of hydrogen bonding between the phosphonate oxygen and the hydrogen of the adjacent amino group. When 1,3-diamines were used as substrates mixtures of products of mono-and di-substitution had been obtained. 31 P NMR spectra revealed interesting non-equivalency of phosphorus atoms of the obtained compounds.
The compounds obtained revealed diverse, albeit promising, activity towards PC-3, MCF-7, and RAW 264.7 cells. Three of them, namely compounds 1, trans-7, and 16, showed broad nonspecific activity, higher than that of incadronic acid (control), influencing the proliferation of all cell lines and thus seem to be of particular interest in the context of anti-osteolytic therapy.
Tetraphosphonate trans-7 was chosen for the medication of sheep with induced osteoporosis, but the result of this therapy was unsatisfactory. This shows that screening done based on the anti-proliferative activity of bisphosphonates towards macrophage-like cell lines is not fully predictive if considering antiosteoporetic activity. On the other hand, medication with bisphosphonates is a long process, while the results of this study were obtained after just two and a half of months of medication with some improvement of the bone status of the sheep observed after next three and a half of months. Thus, this might be seen as a promising result.