Towards a Novel Class of Multitarget-Directed Ligands: Dual P2X7–NMDA Receptor Antagonists

Multi-target-directed ligands (MTDLs) offer new hope for the treatment of multifactorial complex diseases such as Alzheimer’s Disease (AD). Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein—which is open to further optimization—represents the first member of a new class of MTDLs.

Although the pathophysiology of AD is still not completely understood, it is widely accepted that the accumulation of extracellular plaques is one of the major hallmarks in AD [14][15][16][17][18][19]. These plaques are mainly composed of beta-amyloid peptide and activated microglia; in the first instance, they produce a neuroprotective inflammatory response to repair damage [20,21].
The ATP-gated P2X7 receptors (P2X7R), widely expressed in the central nervous system (CNS) [22][23][24], have been recently identified as an obligate participant in the activation of microglia [25], where they are especially abundant. Moreover, its physiological activation in other CNS cell lines-as granular neurons-links to calcium-dependent neuroprotective events such as cAMP response element-binding (CREB) activation [26]; this is responsible for protection against glutamate-induced excitotoxicity and Glycogen synthase kinase 3 (GSK3) phosphorylation, which rescues cells from apoptosis [27]. On top of that, P2X7R activation can also trigger the non-amyloidogenic cleavage of the amyloid precursor protein (APP) [28,29]. On the other hand, with higher and chronic levels of ATP exposure-which is characteristic of AD-the P2X7 receptors up-regulate in microglia [30]. This is responsible for catalyzing inflammatory cascades through the release of IL-1β, ATP, and other factors that lead to cell apoptosis. In addition to this, it has been demonstrated that P2X7R also releases glutamate which activates the ionotropic NMDA receptor [31][32][33].
With physiological levels of glutamate, the NMDA receptor is responsible for learning and memory processes [34]; however, the beta amyloid oligomers that are formed in AD reduce glutamate uptake and increase glutamate release, resulting in an elevation of the neurotransmitter levels in the synaptic cleft [35]. This moderate but chronic stimulation of the NMDA receptor provokes excitotoxicity, neuronal loss, and the characteristic decline in memory and cognition observed in AD patients [36]. The clinically approved NMDA receptor antagonist memantine alleviates this process by blocking the NMDAR in a non-competitive manner [37], allowing the correct function of the receptor while preventing excitotoxicity [38]. Although its usefulness has been proved extensively, this drug cannot totally block the neurotoxic cascade of AD.
Taking into account that tackling this complex disease from only one angle seems to be insufficient, in the last few years several groups have started medicinal chemistry programs aimed at designing multi-target-directed ligands (MTDLs) for hitting different biological targets for AD [39][40][41].
Considering the aforementioned role of NMDA and P2X7 receptors in AD, we reasoned that a drug that could target both receptors simultaneously would be highly beneficial as a potential treatment of this disease; it could not only halt its progression but also ameliorate its symptoms. In addition, the similar double-faced neuroprotective-cytotoxic behavior that both receptors display makes them perfect candidates for this purpose. In light of this, the ideal antagonist would be a compound that displayed inhibitory potencies in the low micromolar range, allowing the physiological function of the receptors but, in turn, inhibiting them upon up-regulation or over-activation.

Results and Discussion
For this, we envisaged the aminoadamantylcarbohydrazides of general structure I as putative dual inhibitors ( Figure 1). Firstly, I features the amantadine scaffold, which is known to be a micromolar NMDAR antagonist (IC 50 = 92 ± 29 µM) [42]; secondly, the N'-arylcarbohydrazide moiety of 1a is a compound endowed with nanomolar P2X7R antagonist activity (IC 50 = 11.6 ± 2 nM) [43]. Taking into account the fact that the introduction of a polar alcohol group in 1a led to a reduction of the antagonist activity of P2X7R (IC 50 = 275 ± 50 nM for 1b) [43], we speculated that the compounds of general structure I might display low micromolar to high nanomolar P2X7R antagonist activities, thus suiting our aim of designing the first dual NMDAR-P2X7R antagonist with low micromolar activities in both targets.
All new compounds were evaluated for their antagonistic effects on 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP)-induced ethidium bromide uptake in human embryonic kidney 293 (HEK 293) cells, stably expressing the human P2X7 receptor, using P2X7 receptor antagonist AZD9056-AstraZeneca's clinical drug candidate [46]-as a positive control. Inspection of the results shown in Table 1 revealed that, with the sole exception of amide 2, all the new compounds showed activities in the micromolar range and only the IC50 value of 9g was determined. The comparison of the IC50 values of 1a, 1b, and 9g (11.6 nM, 275 nM, and 6.0 μM, respectively) highlights that the introduction of a polar group in the adamantane scaffold is deleterious for the P2X7 antagonist activity and that the presence of the basic amino group is even worse than that of the non-basic alcohol group. However, taking into account that we wanted to design a compound endowed with low micromolar inhibitory potencies against both the P2X7 and NMDA receptors, compound 9g suited our purpose.
Additionally, to evaluate whether the synthesized compounds were able to antagonize the NMDA receptors, we measured their effects on the increase in intracellular calcium evoked by NMDA (100 μM, in the presence of 10 μM of glycine) on rat-cultured cerebellar granule neurons [47]. All the compounds were only able to inhibit NMDA in the high micromolar range (see Table S1 in the Supplementary Material), indicating that the addition of the phenyl hydrazide moiety is highly detrimental for NMDA activity. The best compound of the series, 9g, with an IC50 = 468 ± 23 μM, was roughly five-fold less potent than amantadine.
Taking into account the fact that the 3,5-dimethyl derivative of amantadine (i.e., memantine) is 60-fold more potent as an NMDA inhibitor than amantadine [48], we envisaged a second series of derivatives featuring the memantine moiety. Hydrazide 3, featuring two methyl groups in the adamantane scaffold, was reported to be a nanomolar P2X7 inhibitor [44], reinforcing the suitability of this strategy to obtain balanced inhibitory potencies in both receptors. Hence, while the addition of the two methyl groups to the polycyclic scaffold of the adamantane should not be detrimental for P2X7R antagonistic effects, we hoped that their insertion could optimize the activity in the NMDA receptor, leading to a dual micromolar inhibitor of both receptors. Amino acid 11 was prepared using a similar synthetic route, starting from 5,7-dimethyladamantan ecarboxylic acid, 10, but under hardened reaction conditions [45]. After N-protection, coupling with 2-chlorophenylhydrazine or 3,5-dichloro-4-hydrazinopyridine, and deprotection, carbohydrazides 15a-b were obtained (Scheme 2).
All new compounds were evaluated for their antagonistic effects on 2 (3 )-O-(4-benzoylbenzoyl)-ATP (BzATP)-induced ethidium bromide uptake in human embryonic kidney 293 (HEK 293) cells, stably expressing the human P2X7 receptor, using P2X7 receptor antagonist AZD9056-AstraZeneca's clinical drug candidate [46]-as a positive control. Inspection of the results shown in Table 1 revealed that, with the sole exception of amide 2, all the new compounds showed activities in the micromolar range and only the IC 50 value of 9g was determined. The comparison of the IC 50 values of 1a, 1b, and 9g (11.6 nM, 275 nM, and 6.0 µM, respectively) highlights that the introduction of a polar group in the adamantane scaffold is deleterious for the P2X7 antagonist activity and that the presence of the basic amino group is even worse than that of the non-basic alcohol group. However, taking into account that we wanted to design a compound endowed with low micromolar inhibitory potencies against both the P2X7 and NMDA receptors, compound 9g suited our purpose.
Additionally, to evaluate whether the synthesized compounds were able to antagonize the NMDA receptors, we measured their effects on the increase in intracellular calcium evoked by NMDA (100 µM, in the presence of 10 µM of glycine) on rat-cultured cerebellar granule neurons [47]. All the compounds were only able to inhibit NMDA in the high micromolar range (see Table S1 in the Supplementary Material), indicating that the addition of the phenyl hydrazide moiety is highly detrimental for NMDA activity. The best compound of the series, 9g, with an IC 50 = 468 ± 23 µM, was roughly five-fold less potent than amantadine.
Taking into account the fact that the 3,5-dimethyl derivative of amantadine (i.e., memantine) is 60-fold more potent as an NMDA inhibitor than amantadine [48], we envisaged a second series of derivatives featuring the memantine moiety. Hydrazide 3, featuring two methyl groups in the adamantane scaffold, was reported to be a nanomolar P2X7 inhibitor [44], reinforcing the suitability of this strategy to obtain balanced inhibitory potencies in both receptors. Hence, while the addition of the two methyl groups to the polycyclic scaffold of the adamantane should not be detrimental for P2X7R antagonistic effects, we hoped that their insertion could optimize the activity in the NMDA receptor, leading to a dual micromolar inhibitor of both receptors. Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC 50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). Considering that the most promising compound, 9g, in both previous pharmacological evaluations, P2X7R and NMDAR, featured an o-chloro substitution in the phenyl ring, this motif was the one selected for the right-hand side of the structure of our compounds. In addition to this, we decided to prepare 15a due to its similarity with 3 (see Figure 2).
Pleasingly, upon testing with the P2X7R, the IC50 of 15a and 15b were only slightly higher than that of 9g, reinforcing our hypothesis that the introduction of the two-methyl groups would not be deleterious for the P2X7R antagonist activity (Table 1). However, contrary to our expectations, 15a and 15b were unable to significantly inhibit the NMDA receptor even at millimolar concentrations (see Figure 3 and Supplementary Material for details). accumulation assay using hP2X7-expressing human embryonic kidney 293 (HEK293) cells. c Positive control [46]. d IC50 value was obtained from concentration-response curves. e Not active.

Conclusions
To summarize, we have synthesized, characterized and evaluated a series of novel adamantylcarbohydrazides as potential dual NMDAR and P2X7R antagonists. While three derivatives displayed low micromolar activities as P2X7R antagonists, only 9g showed some activity as an NMDA receptor antagonist. Compound 9g is, to the best of our knowledge, the first dual antagonist of both receptors.

General Methods
Melting points were determined in open capillary tubes with an MFB 595010 M Gallenkamp. A Varian Mercury 400 spectrometer recorded 400 MHz 1 H-NMR spectra and 100.6 MHz 13 C-NMR spectra. The chemical shifts are reported in ppm (δ scale) relative to internal tetramethylsilane, and coupling constants are reported in hertz (Hz). Assignments given for the NMR spectra of the new compounds have been carried out on the basis of COSY 1 H/ 13 C (gHSQC and gHMBC sequences), COSY 1 H/ 1 H (standard procedures). IR spectra were run on PerkinElmer Spectrum RX I spectrophotometer (Waltham, MA, USA). Absorption values are expressed as wavenumbers (cm −1 ); only significant absorption bands are given. The GC/MS analysis was carried out in an inert Agilent Technologies 5975 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an Agilent 122-5532 DB-5MS 1b (30 m × 0.25 mm) capillary column with a stationary phase of phenylmethylsilicon (5% diphenyl-95% dimethylpolysiloxane), using the following conditions: initial temperature of 50 °C (1 min), with a gradient of 10°C/min up to 300 °C, and a temperature in the source of 250 °C, solvent delay (SD) of 4 min, and a pressure of 7.35 psi. Accurate mass measurements were obtained using the ESI technique. Absorption thin-layer chromatography was performed with aluminum-backed sheets with silica gel 60 F254 (Merck, Darmstadt, German, ref 1.05554), and spots were visualized with UV light and 1% aqueous solution of KMnO4. 4.1.2. Synthesis of the Starting Acids 6 and 12 [45] 3-acetamidoadamantane-1-carboxylic acid. The compound 1-Adamantane carboxylic acid, 4, (5.0 g, 27.7 mmol) was suspended in 70% HNO3 (4.0 mL) and cooled at 0 °C with an ice bath. Using an addition funnel, 98% H2SO4 (30 mL) was added dropwise, at such a rate that the temperature was kept at 0 °C or below. The reaction mixture was stirred for 2 h at 0 °C, whereupon acetonitrile (20 mL) was added via an addition funnel, at such a rate that the reaction mixture was kept at 0 °C. After

Conclusions
To summarize, we have synthesized, characterized and evaluated a series of novel adamantylcarbohydrazides as potential dual NMDAR and P2X7R antagonists. While three derivatives displayed low micromolar activities as P2X7R antagonists, only 9g showed some activity as an NMDA receptor antagonist. Compound 9g is, to the best of our knowledge, the first dual antagonist of both receptors. 3-Acetamidoadamantane-1-carboxylic acid. The compound 1-Adamantane carboxylic acid, 4, (5.0 g, 27.7 mmol) was suspended in 70% HNO 3 (4.0 mL) and cooled at 0 • C with an ice bath. Using an addition funnel, 98% H 2 SO 4 (30 mL) was added dropwise, at such a rate that the temperature was kept at 0 • C or below. The reaction mixture was stirred for 2 h at 0 • C, whereupon acetonitrile (20 mL) was added via an addition funnel, at such a rate that the reaction mixture was kept at 0 • C. After all the acetonitrile was added, the reaction was stirred at 0 • C for 3 h. The reaction mixture was then poured over ice (ca. 200 g), with shaking, and was allowed to warm to room temperature. The colorless solid was then filtered over vacuum, washed with water, and dried overnight to give the title compound (5.8 g, 88.1% yield). The 1 H-NMR matched the data previously reported in the bibliography [46].

Chemical Synthesis
3-Aminoadamantane-1-carboxylic acid hydrochloride, 5·HCl. The compound 3-acetamidoadamantane-1-carboxylic acid (5.8 g, 24.4 mmol), water (13.5 mL), and 37% hydrochloric acid (33.0 mL) were added to a 3-neck round bottom flask equipped with a reflux condenser, thermometer, and a mechanical stirrer; the mixture was heated at reflux for 6 days. After cooling at 0 • C with an ice bath, the white precipitate solid was carefully filtered. The solid obtained was then dried under vacuum for 1 day to afford 5·HCl (2.6 g, 45.9% yield). The 1 H-NMR matched the data previously reported in the bibliography [46].
3-[(tert-Butoxycarbonyl)amino]adamantane-1-carboxylic acid, 6. Aqueous 2 M NaOH (10 mL) and di-tert-butyl dicarbonate (2.52 g, 11.7 mmol) were added to a mixture of 5·HCl (2.6 g, 11.2 mmol) in THF (26 mL). After stirring for 16 h at room temperature, the reaction was cooled in an ice bath. Upon neutralization with aqueous 2 M HCl, the mixture was partitioned into ethyl acetate and water. The organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure to give impure 6 (2.66 g). After purification by crystallization from acetonitrile, 6 (1.5 g, 45.3% yield) was obtained as a white solid. The 1 H-NMR matched the data previously reported in the bibliography [46].
3-Acetamido-5,7-dimethyladamantane-1-carboxylic acid. The compound 3,5-dimethyladamantane -1-carboxylic acid (2.0 g, 9.6 mmol) was suspended in 70% HNO 3 (6.0 mL) and cooled to 0 • C with an ice bath. Using an addition funnel, 98% H 2 SO 4 (10 mL) was added dropwise at such a rate that the temperature was kept at 0 • C or below. The mixture was stirred for 10 min and 20-30% oleum (3.5 mL) was added dropwise. The whole mixture was stirred for 30 min at 0 • C and 1 h at room temperature. Upon cooling again at 0 • C, acetonitrile (6.0 mL) was added and the mixture stirred for 10 min at 0 • C and 3 h at room temperature. Finally, the mixture was poured onto ice (ca. 200 g) and kept at 5 • C overnight. The white precipitate was collected via suction filtration and dried for 1 h to obtain the title compound as a white solid in quantitative yield [46].
3-Amino-5,7-dimethyladamantane-1-carboxylic acid hydrochloride, 11·HCl. The compound 3-acetamido -5,7-dimethyladamantane-1-carboxylic acid (2.5 g, 9.42 mmol) was heated in 37% HCl (60 mL) for 5 days at reflux in a round flask equipped with a reflux condenser, thermometer, and a mechanical stirrer. After removal of the acid under reduced pressure, the crude product was washed with ethyl acetate and collected via suction filtration. The compound was dried in a desiccator over P 2 O 5 overnight to give 11·HCl (1.23 g, 50% yield) as a white solid. The 1 H NMR matched the data previously reported in the bibliography [45].
3-tert-Butoxycarbonylamino-5,7-dimethyladamantane-1-carboxylic acid, 12. The compound Di-tert-butyl dicarbonate (1.03 g, 4.73 mmol) was added to a round flask containing 11·HCl (1.23 g, 4.73 mmol) in THF (15 mL) and aqueous 2 M NaOH (6 mL). After stirring for 16 h at room temperature, the reaction was cooled into an ice bath. Following this, it was neutralized with aqueous 2 M HCl and partitioned into ethyl acetate and water. The organic layer was separated, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a white solid (1.15 g). The crude product was purified by crystallization from acetonitrile to give 12 (800 mg, 52% yield). The 1 H-NMR matched the data previously reported in the bibliography [45] General Procedure A. HOBt (162 mg, 1.2 mmol) and EDC (230 mg, 1.2 mmol) were added to a suspension of 6 (295 mg, 1 mmol) or 12 (323 mg, 1 mmol) in CH 2 Cl 2 and the whole mixture was stirred at room temperature for 2 h. The resulting mixture was then added dropwise via an addition funnel to a solution of the desired hydrazine (2 mmol, see each example for specific weights) in CH 2 Cl 2 , maintaining the temperature range between 0-10 • C. The mixture was washed with water and aqueous K 2 CO 3 , and the organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure, to yield the corresponding carbamate 8a-f or 14a-b.
General Procedure B. A solution of the corresponding crude carbamate precursor 8a-f or 14a-b (ca. 1 mmol) in CH 2 Cl 2 (20 mL) had 4 M HCl in 1,4-dioxane (10 mL) added to it; the solution was left stirring at room temperature for 16 h. Once the reaction was completed, the mixture was extracted with aqueous 1 M HCl and the aqueous phase was then brought to basic pH with excess of aqueous 2 M NaOH. Upon extraction with CH 2 Cl 2 , the joined organic extracts were dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude reaction product was crystallized from ethyl acetate, to yield the desired pure product 9a-f or 15a-b. Data for 2, 9a-f, and 15a-b. 3-Amino-N-(2-chlorobenzyl)adamantane-1-carboxamide, 2. Following general procedure A but using 2-chlorobenzylamine (283 mg, 2 mmol) instead of a hydrazine, the expected carbamate that was obtained was subsequently treated with the general procedure B to yield 2 as a white solid (111 mg, 35% overall yield). mp = 186-189 3-Amino-N'-(2,6-dichlorophenyl)adamantane-1-carbohydrazide, 9b.