Transformation of Pro-Leu-Gly-NH₂ Peptidomimetic Positive Allosteric Modulators of the Dopamine D₂ Receptor into Negative Modulators

Abstract

The synthesis of dimethyl derivatives of 5.6.5 spiro bicyclic lactam Pro-Leu-Gly-NH₂ peptidomimetics was carried out to test the hypothesis that by placing methyl groups on the β-methylene carbon of the thiazolidine ring steric bulk would be introduced into the topological space that the β-methylene carbon is believed to occupy in the negative allosteric modulators of the dopamine D₂ receptor. With such a modification, a positive allosteric modulator would be converted into a negative allosteric modulator. This hypothesis was shown to be correct as 3a and 4a where found to be negative allosteric modulators, whereas their unmethylated derivatives were positive allosteric modulators of the dopamine D₂ receptor.

The endogenous neuropeptide l-prolyl-l-leucyl-glycinamide (PLG) is a positive allosteric modulator of dopamine receptors.¹⁻³ PLG and its peptidomimetics show selectivity toward the D_2S, D_2L, and D₄ dopamine receptor subtypes.^1,4 They do not exhibit activity at the D₁ dopamine receptor or the closely related adrenergic receptors.^1,4 PLG and its peptidomimetics exert their positive modulatory effects by increasing the affinity of the high-affinity state of the dopamine receptor for orthosteric agonists and by increasing the ratio of the dopamine receptor in the high-affinity state, which is coupled to G-proteins.^1,4 In vivo, PLG and its peptidomimetics enhance both amphetamine and apomorphine-dependent rotational behavior in 6-hydroxydopamine-lesioned rats,⁵⁻⁷ prevent and reverse drug-induced dopamine receptor supersensitivity caused by dopamine receptor antagonists,^8,9 and inhibit neuroleptic drug-induced vacuous chewing in rat models of tardive dyskinesia.^7,10

The synthesis of conformationally constrained analogues of PLG led to highly active PLG peptidomimetics such as 1a (Figure 1), which possesses the very rigid 5.6.5 spiro bicyclic lactam type II β-turn mimic.¹¹ These studies provided strong support for the hypothesis that the bioactive conformation of PLG is a type II β-turn. However, the dopamine receptor modulating activity of triproline Pro-Pro-Pro-NH₂ did not fit this hypothesis, since it cannot exist in a type II β-turn.¹² We hypothesized that Pro-Pro-Pro-NH₂ was adopting a different conformation that placed the important pharmacophore groups in relatively the same topographical space as did the PLG peptidomimetics designed to mimic a type II β-turn. The synthesis of Pro-Pro-Pro-NH₂ peptidomimetic 2a possessing a highly constrained 5.6.5 spiro bicyclic lactam scaffold designed to mimic a polyproline II helix was carried out to test this hypothesis.¹³

Figure 1.

PLG peptidomimetics based upon the 5.6.5 spiro bicyclic scaffold.

Peptidomimetics 1a and 2a were found to be positive allosteric modulators of the dopamine receptor, thus providing support for the concept that conformationally different peptidomimetics are capable of serving as allosteric modulators of the dopamine receptor so long as these conformationally constrained scaffolds place key pharmacophores in the correct region of space. In a novel finding, however, the bridgehead epimers of 1a and 2a, peptidomimetics 1b and 2b, respectively, were found to be negative modulators of the dopamine receptor.¹³ Since the structural difference between 1a and 1b and between 2a and 2b is the chirality of the bridgehead carbon atom, we postulated that the difference in bridgehead chirality has an effect on the conformational nature of the thiazolidine portion of the spiro bicyclic lactam scaffold that in turn translated into different conformational changes at the PLG allosteric site upon binding thereby bringing about different modulatory effects. In particular, modeling studies suggested that the pucker of the thiazolidine ring in the scaffolds of 1b and 2b caused the β-methylene carbon to jut into a different area of topological space than that found in 1a and 2a (Figure 2A).

Figure 2.

(A) Overlay of the 5.6.5 spiro bicyclic lactam scaffolds of 2a and 2b. (B) Overlay of 2a and 4a. The lowest energy conformers were obtained with the Maestro 9.1 Suite developed by Schrodinger. In particular, the OPLS force field was selected in Macromodel, which was initially used to conduct the conformational analysis. This was followed by conducting an ab initio QM energy optimization with Jaguar. The optimizations were performed on each of the lowest energy conformers obtained using Macromodel. All calculations were performed on the structurally intact PLG peptidomimetic; any structural features missing were removed for the purpose of clarity.

In this study, we have synthesized the dimethyl derivatives of 1a, 1b, 2a, and 2b, compounds 3a, 3b, 4a, and 4b, respectively, to test the above hypothesis. By placing methyl groups on the β-methylene carbon of the thiazolidine ring, we have attempted to place steric bulk in the topological space that the β-methylene carbon occupies in its different pucker conformations (Figure 2B). We hypothesize that this type of substitution will convert a positive allosteric modulator into a negative allosteric modulator.

Results and Discussion

Syntheses

The synthesis of the 5.6.5. spiro bicyclic lactam PLG peptidomimetics 3a, 3b, 4a, and 4b were carried out as outlined in Schemes 1 and 2. α-Substituted proline 5 served as the starting material for the synthesis of these peptidomimetics. This compound was synthesized as described previously by us.^11,13,14 Compound 5 was condensed in separate reactions with the hydrochloride salts of l- and d-penicillamine methyl esters to give the respective thiazolidines 6a and 6b as diastereoisomeric mixtures. The immediate lactamization of 6a and 6b in the presence of 2-chloro-1-methyl pyridinium iodide (CMPI) gave the corresponding pairs of 5.6.5-spiro bicyclic thiazolidines scaffolds 7a/7b and 8a/8b in overall yields of 63% and 57%, respectively. For the condensation/lactamization sequence with the l-penicillamine derivative, a 3:1 ratio of the two C-8a′ epimers 7a and 7b was obtained with the major diastereoisomer 7a having the R configuration at C-8′a (bridgehead hydrogen down). In contrast, in the reaction sequence with d-penicillamine-OMe, a 1:1 ratio of the diastereoisomers 8a and 8b was obtained. These diastereoisomeric ratios directly mirrored the diastereoselectivities observed previously with condensations carried out with d- and l-cysteine-OMe.^11,13 The absolute and relative stereochemical configuration about the bridgehead carbons (C-8′a) for each diastereoisomer was established by 1-D NOE experiments, as well as by single molecule X-ray structure analysis for three of the four diastereoisomers (see Supporting Information for structures of compounds 7b, 8a, and 8b).¹⁵

Scheme 1.

Scheme 2.

All four diastereoisomers derived from the penicillamines, 7a, 7b, 8a, and 8b, were separately N-deprotected in the presence of TFA in anhydrous CH₂Cl₂ and subsequently coupled to Boc-l-Pro-OH in the presence of HATU as an activating agent to afford 9a, 9b, 10a, and 10b, respectively. We initially tried to convert 9b to the carboxamide via ammonolysis, but this was unsuccessful as complete recovery of the starting material was obtained even when the ammonolysis was carried out under forcing conditions. Since our previous experience with the l-cysteine-OMe derivative did not present such problems,¹³ we suspect that the added steric bulk of the 2′-gem-dimethyl groups hinders the nucleophilic attack on the ester carbonyl. We therefore decided to use the ester hydrolysis/ammonia coupling protocol that had been successfully used before.¹³ Ester hydrolysis of 9b proceeded smoothly under standard conditions without any epimerization. The subsequent coupling of the acid with ammonia resulted in a 20–25% yield of the carboxamide 11. Despite the poor yield, the final N-deprotection was conducted with TFA/CH₂Cl₂ to give the trifluoroacetate salt of 4b in quantitative yield.

Unfortunately, diastereoisomer 9a was recalcitrant toward ester hydrolysis under standard conditions and required almost 10 equiv of LiOH, refluxing conditions, and prolonged reaction times for complete conversion. Under these forced conditions, almost complete epimerization at the C-3′ position (as determined by NOE analysis) was observed. Although epimerization under ester hydrolysis had not been observed for the previously synthesized des-dimethyl derivative, there is precedent for C-3′ epimerization under forced ammonolysis conditions.¹³ We postulate that the pseudoaxial positioning of the methoxycarbonyl moiety in 9a, in addition to the C-2′-gem-dimethyl groups, hinders ester hydrolysis and reinforces the epimerization under the harsh conditions. The carboxylic acid obtained was subjected to amidation via coupling with ammonia with HATU to give carboxamide 12. For diastereoisomer 10a, ester hydrolysis was quite facile with carboxylic acid formation complete and without epimerization within 6–8 h with 3 equiv of LiOH.¹¹ The carboxylic acid was coupled with ammonia to form carboxamide 12. Removal of the Boc-group from 12 with TFA/CH₂Cl₂ gave the corresponding TFA salt of 3a as a crystalline white solid.

In light of the epimerization issues encountered above, we tried to introduce the carboxamide moiety earlier within the synthetic scheme by attempting to carry out the ammonolysis on the diastereoisomeric thiazolidine mixture 6a/6b. Unfortunately, the ammonolysis reaction was unsuccessful even under forced reaction conditions. We next attempted utilization of either the d- or l-penicillamine carboxamide in the condensation/lactamization sequence. An attempt to directly synthesize d-penicillamine carboxamide by ammonolysis of d-Pen-OMe in anhydrous MeOH failed. However, by masking the amine and thiol functionalities through the synthesis of the C-2 phenyl thiazolidine derivatives^16,17d- and l-penicillamine were converted to d- and l-penicillamine carboxamide, 13a and 13b, respectively (see Supporting Information for the synthetic details).

Compounds 13a and 13b were utilized in the formation of the 5.6.5-spiro bicyclic lactam peptidomimetics 3b and 4a, respectively, as depicted in Scheme 2. The condensation of 13a with 5 provided 14a as a mixture of diastereoisomers. Mukaiyama′s reagent, 2-chloro-1-methylpyridinium iodide, facilitated the cyclization of these diastereoisomeric thiazolidines to give a mixture of diastereoisomers 15a and 15b in a 2:5 ratio and in an overall yield of 71%. These diastereoisomers were separable by column chromatography and a crystal structure was obtained for 15b (see Supporting Information for structure).¹⁵ Isomer 15b was treated with TFA to remove the tert-butoxycarbonyl group and the resulting amine was coupled with Boc-l-Pro-OH in the presence of HATU and DIPEA to give 16. N-Deprotection of 16 gave 3b as its trifluoroacetate salt.

In a similar manner, 13b was condensed with 5 to give 14b, the cyclization of which with Mukaiyama′s reagent gave an inseparable mixture of diastereoisomers 17. The removal of the tert-butoxycarbonyl group from 17 with TFA and the subsequent coupling of the amine to Boc-l-Pro-OH with HATU and DIPEA yielded diastereoisomers 11 and 18 in a 1:2 ratio, which were separated by silica gel column chromatography. The stereochemistry at the bridgehead carbon (C-8′a) of the bicyclic thiazolidine lactams was assigned on the basis of extensive NOE difference experiments. Removal of the tert-butoxycarbonyl protecting group from 18 with TFA/CH₂Cl₂ provided the desired PLG peptidomimetic 4a as its trifluoroacetate salt.

Pharmacological Studies

The PLG peptidomimetics 3a, 3b, 4a, and 4b were evaluated for their ability to increase or decrease the binding of tritiated N-propylnorapomorphine ([³H]NPA) on bovine striatal dopamine D₂ receptors. The percentage change in total specific [³H]NPA binding was measured across various concentrations (10^–5–10^–10 M) of each peptidomimetic. The results are depicted in Figure 3. Peptidomimetics 3b and 4b showed negative modulatory effects in their ability to affect [³H]NPA binding to isolated dopamine D₂ receptors, but at no concentrations were the effects significant. In contrast, 3a and 4a significantly decreased agonist binding to dopamine D₂ receptors, displaying a dose-dependent effect. Peptidomimetic 3a produced its maximum effect at 10^–6 M where it decreased the binding of [³H]NPA by 14.8 ± 1.9%. Peptidomimetic 4a showed significant effects at concentrations of 10^–7, 10^–6, and 10^–5 M with the maximal effect exhibited at 10^–5 M. At this concentration, the binding of [³H]NPA to isolated dopamine D₂ receptors was decreased by 15.4 ± 2.0%.

Figure 3.

Modulation of [³H]NPA binding to bovine striatal dopamine D₂ receptors by PLG peptidomimetics 3a, 3b, 4a, and 4b. Data represents the percent increase in specific [³H]NPA binding relative to the control value when the indicated concentration of peptidomimetic was added directly to the assay buffer. Results are the mean ± SEM of at least six separate experiments carried out in triplicate. The data were analyzed by a one way analysis of variance followed by a Dunnett’s post hoc test: (*) significantly different (p < 0.05) from the control value; (**) significantly different (p < 0.01) from the control value.

Concentration response curves were generated to obtain EC₅₀ values for [³H]NPA at bovine striatal dopamine D₂ receptors in the presence and absence of peptidomimetics 3a and 4a. These results are summarized in Table 1. Under control conditions, the EC₅₀ for [³H]NPA binding was 0.41 ± 0.055 nM. When 3a, at a concentration of 10^–6 M, was added to the dopamine receptor preparation, the EC₅₀ of [³H]NPA increased significantly to 1.12 ± 0.35 nM. This represented a 2.7-fold shift in the EC₅₀ value. A similar effect was seen with 10 μM 4a. In this case, the EC₅₀ of [³H]NPA was increased to a value of 1.15 ± 0.37 nM, which represented 2.8-fold shift. Thus, both 3a and 4a decreased the affinity of the agonist NPA to dopamine D₂ receptors.

Table 1. Effect of 3a and 4a on [³H]NPA’s EC₅₀ Value at Dopamine D₂ Receptors^a.

treatment (conc.)	EC50 [3H]NPA (nM)	EC50 fold-shift
control (H2O)	0.41 ± 0.055
3a(1 μM)	1.12 ± 0.35b	2.7
4a(10 μM)	1.15 ± 0.37b	2.8

^aThe results are the mean of at least four separate experiments each carried out in triplicate. The data were analyzed by a one way analysis of variance followed by an unpaired t test.

^bp < 0.01 compared to control.

On the basis of the difference in allosteric modulatory activity of the dopamine receptor between the peptidomimetics 1a and 2a and 1b and 2b, we postulated that the difference in bridgehead chirality between these pair of compounds has an effect on the puckering conformations of the thiazolidine ring of the spiro bicyclic lactam scaffold wherein moieties like the β-methylene carbon jut into a different area of topological space in 1b and 2b than that seen in 1a and 2a. This in turn translates into different conformational changes at the PLG allosteric site upon binding, thereby bringing about opposite modulatory effects.

In this study, the dimethyl derivatives of 1a, 1b, 2a, and 2b, compounds 3a, 3b, 4a, and 4b, respectively, were synthesized to test this hypothesis. It was felt that by placing methyl groups on the β-methylene carbon of the thiazolidine ring of the positive modulators 1a and 2a steric bulk would be introduced into the topological space that the β-methylene carbon was believed to occupy in the negative allosteric modulators 1b and 2b. If our hypothesis was correct, we expected that this type of substitution would convert the positive allosteric modulators into negative allosteric modulators. This is what was observed. The dimethyl derivatives of the positive allosteric modulators 1a and 2a, peptidomimetics 3a and 4a, became a negative modulators of the dopamine receptor. This indicates that the introduction of dimethyl groups into the structure of the positive modulators is able to mimic the structural/conformational characteristics of the unsubstituted negative modulators in terms of interacting with the allosteric binding site.

In contrast, when dimethyl groups are placed on the thiazolidine β-methylene carbon of the dopamine receptor negative allosteric modulators 1b and 2b to give peptidomimetics 3b and 4b, the resulting compounds retain their negative modulatory activity, but only at a weak level. A comparison of 2band 4b, for example, shows that the thiazolidine β-methylene carbons of these two compounds overlay quite well. This means that the methyl groups of 4b occupy topographical space beyond that occupied by the thiazolidine β-methylene carbon of 2b. We postulate that the steric bulk of the methyl groups in this case creates structural/conformational changes at the allosteric site that are less conducive to negatively modulating the dopamine receptor.

In conclusion, this study demonstrates that small structural changes in these spiro bicyclic dopamine receptor modulators can bring about major changes in modulatory activity of the compounds. We are presently investigating whether both methyl groups are responsible for these dramatic effects through the synthesis of the monosubstituted derivatives of 3a, 3b, 4a, and 4b.

Methods

Compound Purity

The purity of compounds 3a, 3b, 4a, and 4b was >95% as determined by HPLC.

Methyl [3′R-(3′β,6′α,8′aα)]-1-(tert-Butyloxycarbonyl)-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (7a) and Methyl [3′R-(3′β,6′α,8′aβ)]-1-(tert-Butyloxycarbonyl)-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (7b)

To a chilled (ice/water/salt) solution of the α-substituted proline derivative 5 (1.9 g, 7.0 mmol) in 1:1 95% EtOH/H₂O was added solid NaHCO₃ (588 mg, 7.0 mmol). l-Penicillamine methyl ester hydrochloride (1.4 g, 7.0 mmol) was then added as a solid in one portion. The suspension eventually formed a mostly clear solution, the pH of which was adjusted to 6.5–7.0 with sat. NaHCO₃ solution. It was then stirred overnight under ambient conditions. The next morning EtOH was removed in vacuo and the pale pink-colored aqueous solution was acidified to pH 4–5. The resulting emulsion was extracted using a 3:1 mixture of Et₂O/EtOAc (3×). The organic layers were pooled together, dried over MgSO₄, filtered, and concentrated in vacuo to give a pale pink oil that turned into a pink-colored foam when placed under vacuum. The crude mixture of diastereoisomers 6a (2.1 g, 70% yield) was directly subjected to the lactam formation reaction below.

The diastereomeric thiazolidine mixture 6a (2.1 g, 5.0 mmol) was dissolved in 150 mL anhydrous CH₂Cl₂. Triethylamine (1.7 mL, 12.5 mmol) was added in one portion to the solution followed by CMPI (1.5 g, 6.0 mmol). A bright yellow-colored suspension was obtained. This suspension was heated at reflux for 17 h and at the end of this time a dark amber-colored solution was obtained. The reaction mixture was washed sequentially with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated to give an orange-colored syrup that was purified by flash chromatography (gradient elution: 10–66% EtOAc in hexanes; very slow increments in polarity and using a column diameter that would be used for a 7–8 g reaction scale). The two diastereoisomers, 7a and 7b, were obtained in a 3:1 ratio and in an overall yield of 63%.

7a

Oil (900 mg); TLC Rf 0.40 (EtOAc/hexanes, 1:1); [α]_D +13.7 (c 0.75, CH₂Cl₂). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.11 (dd, J₁ = 11.2 Hz, J₂ = 3.4 Hz, 0.4H, 8′a-CH), 4.97 (dd, J₁ = 11.4 Hz, J₂ = 3.4 Hz, 0.6H, 8′a-CH), 4.37 and 4.31 (s, 1H, 3′-CH), 3.68 and 3.67 (s, 3H, OCH₃), 3.57–3.40 (m, 2H, 5-CH₂), 2.61 (ddd, J₁ = J₂ = 13.7 Hz, J₃ = 3.1 Hz, 0.4H, 3-CH₂), 2.37 (m, ddd, J₁ = J₂ = 13.7 Hz, J₃ = 2.6 Hz, 0.6H, 7′-CH₂), 2.29–2.14 (m, 2H, 8′-CH₂, 3-CH₂), 2.02–1.71 (m, 5H, 8′-CH₂, 7′-CH₂, 3-CH₂, 4-CH₂), 1.61 and 1.57 (s, 3H, 2′-C(CH₃)₂), 1.38 (s, 9H, C(CH₃)₃), 1.34 and 1.32 (s, 3H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃, gHMQC) δ 171.6 and 171.4 (lactam C=O), 170.0 and 169.8 (ester C=O), 153.9 and 153.3 (carbamate C=O), 80.1 and 79.5 (C(CH₃)₃), 72.2 and 71.8 (3′-C), 64.8 and 64.7 (8′a-C), 62.3 and 62.0 (2′-C), 52.1 and 51.9 (OCH₃), 51.3 and 51.2 (6′-C), 48.7 and 48.4 (5-C), 39.6 and 38.8 (8′-C), 33.1 and 32.3 (7′-C), 32.2 and 31.9 (2′-C(CH₃)₂), 28.7and 28.6 (C(CH₃)₃), 27.2 and 27.1 (3-C), 24.6 and 24.5 (2′-C(CH₃)₂), 23.4 and 22.8 (4-C); ESI HRMS m/z 421.1781 [M + Na]⁺, (C₁₉H₃₀N₂O₅S + Na⁺) requires 421.1768.

7b

White solid (300 mg); suitable crystals for an X-ray structure¹⁵ were obtained after crystallization from hexanes and EtOAc; mp 160–162 °C (dec); TLC Rf 0.42 (EtOAc/hexanes, 1:1); [α]_D −198.5 (c 0.4, CH₂Cl₂). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.08 (dt, J₁ = 10.2 Hz, J₂ = 3.1 Hz, 1H, 8′a-CH), 4.75 and 4.63 (s, 1H, 3′-CH), 3.72 and 3.71 (s, 3H, OCH₃), 3.64–3.38 (m, 2H, 5-CH₂), 2.58–1.75 (m, 8H, 7′-CH₂, 8′-CH₂, 3-CH₂, 4-CH₂), 1.60 and 1.59 (s, 3H, 2′-C(CH₃)₂), 1.41 (app. s, 12H, C(CH₃)₃, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃, gHMQC assignment) δ 171.2 and 170.8 (lactam C=O), 169.2 and 169.0 (ester C=O), 154.4 and 154.0 (carbamate C=O), 80.4 and 79.3 (C(CH₃)₃), 72.1 and 71.8 (3′-C), 63.9 and 63.5 (2′-C), 62.7 and 62.3 (8′a-C), 52.2 and 52.1 (6′-C), 51.1 (OCH₃), 48.6 and 48.4 (5-C), 43.2 and 42.2 (3-C), 36.0 and 35.8 (7′-C), 28.9, 28.8, 28.7, 28.6, 28.5 (C(CH₃)₃, 2′-C(CH₃)₂), 27.8 and 26.9 (8′-C), 22.9 and 22.4 (4-C); ESI HRMS m/z 421.1755 [M + Na]⁺, (C₁₉H₃₀N₂O₅S + Na⁺) requires 421.1768.

Methyl [3′S-(3′α,6′α,8′aα)]-1-(tert-Butyloxycarbonyl)-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (8a) and Methyl [3′S-(3′α,6′ α,8′aβ)]-1-(tert-Butyloxycarbonyl)-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (8b)

For the synthesis of the diastereoisomeric mixture 6b, compound 5 was treated with commercially available d-penicillamine methyl ester hydrochloride in the same manner as that described above for synthesis of 6a. The crude diastereomeric thiazolidine mixture 6b(1.9 g, 4.7 mmol) was then treated in the same manner as that describe above for the compound 6a. The two diastereoisomers 8a and 8b were obtained in a 1:1 ratio and in an overall yield of 57%.

8a

Yield 500 mg; suitable crystals for an X-ray structure¹⁵ were obtained after crystallization from hexanes and EtOAc; mp 166–167 °C (dec); TLC Rf 0.34 (EtOAc/hexanes, 1:1); [α]_D +138.4 (c 0.5, CH₂Cl₂). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.32 (dd, J₁ = 10.8 Hz, J₂ = 3.9 Hz, 0.4H, 8′a-CH), 5.23 (dd, J₁ = 11.0 Hz, J₂ = 4.2 Hz, 0.6H, 8′a-CH), 4.61 and 4.57 (s, 1H, 3′-CH), 3.76 and 3.71 (s, 3H, OCH₃), 3.68–3.44 (m, 2H, 5-CH₂), 2.79–2.64 (m, 1H, 7′-CH₂), 2.32–2.20 (m, 2H, 8′-CH₂, 3-CH₂), 2.04–1.71 (m, 5H, 8′-CH₂, 7′-CH₂, 3-CH₂, 4-CH₂), 1.59 and 1.56 (s, 3H, 2′-C(CH₃)₂), 1.49–1.42 (m, 12H, 2′-C(CH₃)₂, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC assignment) δ 171.7 and 171.0 (lactam C=O), 169.0 and 168.7 (ester C=O), 153.6 and 153.5 (carbamate C=O), 80.5 and 79.3 (C(CH₃)₃), 72.3 and 72.1 (3′-C), 64.6 and 64.5 (2′-C(CH₃)₂), 62.4 and 62.3 (8′a-C), 51.9 and 51.8 (OCH₃), 51.4 and 51.1 (6′-C), 48.6 (5-C), 41.0 and 38.9 (3-C), 32.5 and 31.5 (7′-C), 30.3 and 30.0 (2′-C(CH₃)₂, 28.9, 28.5, 28.2, 27.8 (8′-C and C(CH₃)₃), 27.1 and 26.6 (4-C); ESI HRMS m/z 421.1792 [M + Na]⁺, (C₁₉H₃₀N₂O₅S + Na⁺) requires 421.1768.

8b

Yield 500 mg; suitable crystals for an X-ray structure¹⁵ were obtained after crystallization from hexanes and EtOAc; mp 163–164 °C (dec); TLC Rf 0.26 (EtOAc/hexanes, 1:1); [α]_D −130.5 (c 0.5, CH₂Cl₂). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 4.93 (dd, J₁ = 11.1 Hz, J₂ = 2.1 Hz, 1H, 8′a-CH), 4.27 and 4.26 (s, 1H, 3′-CH), 3.74 and 3.69 (s, 3H, OCH₃), 3.67–3.55 (m, 1H, 5-CH₂), 3.52–3.39 (m, 1H, 5-CH₂), 2.68–2.57 (m, 1.2H, 7′-CH₂), 2.36–2.25 (m, 0.8H, 8′-CH₂), 2.19–1.74 (m, 6H, 8′-CH₂, 7′-CH₂, 3-CH₂, 4-CH₂), 1.59 (s, 3H, 2′-C(CH₃)₂), 1.48–1.38 (m, 12H, 2′-C(CH₃)₂, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC assignment) δ 172.3 and 171.8 (lactam C=O), 169.8 and 169.7 (ester C=O), 154.4 and 154.1 (carbamate C=O), 80.5 and 79.1 (C(CH₃)₃), 73.5 and 73.4 (3′-C), 72.4 and 72.3 (6′-C), 63.9 and 63.6 (8′a-C), 61.5 and 61.47 (2′-C(CH₃)₂), 52.0 and 51.9 (O–CH₃), 48.3 (5-C), 44.6 and 42.9 (3-C), 36.7 (7′-C), 31.8 and 31.7 (2′-C(CH₃)₂), 28.9 and 28.5 (8′-C), 28.3 and 28.28 (C(CH₃)₃), 23.5 and 23.4 (2′-C(CH₃)₂), 22.7 and 22.0 (4-C); ESI HRMS m/z 421.1778 [M + Na]⁺, (C₁₉H₃₀N₂O₅S + Na⁺) requires 421.1768.

Methyl [3′R-(3′β,6′α,8′aα)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (9a)

A solution of the spiro bicyclic compound 7a (500 mg, 1.3 mmol) in 11 mL of anhydrous CH₂Cl₂ was cooled to 0 °C in an ice bath. Trifluoroacetic acid (3.8 mL, 50 mmol) was then added dropwise to this solution which was then stirred at 0 °C until removal of the Boc-group was determined to be complete by TLC. This was usually between 1.5 and 2 h. The reaction was warmed up to rt, and the volatiles removed by rotary evaporation. The residue was redissolved in CH₂Cl₂ and the solvent removed by evaporation twice until the excess TFA was removed. The pale yellow-colored residue was dried to a constant weight for 5–6 h under vacuum.

A solution of Boc-l-proline (437 mg, 2.0 mmol) in 5 mL of DMF was cooled to −5 °C in an ice/salt bath. DIPEA (0.6 mL, 3.4 mmol) was added dropwise to this cooled solution followed by HATU (772 mg, 2.0 mmol). The clear solution was stirred for 30 min after which a solution of the spirocycle free amine in 5 mL of DMF [obtained by neutralizing the TFA salt (700 mg, 1.7 mmol) with DIPEA (0.6 mL, 3.4 mmol)] was added to the active ester. The yellow-colored solution was warmed to rt and then stirred under Ar for 36 h. At the end of this time, DMF and other volatiles were removed in vacuo. The residue was dissolved in 80 mL of EtOAc, and this solution was sequentially washed with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated to give a yellow-colored residue which was purified by flash chromatography (gradient elution: 0–6% MeOH/CH₂Cl₂ in discrete steps) to give a clear oil weighing 492 mg (79% yield). TLC Rf 0.47 (CH₂Cl₂/MeOH, 20:1); [α]_D −33 (c 1.11, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.17 (dt, 1H, J₁ = 11.0 Hz, J₂ = 3.0 Hz, 8′a-CH), 4.44 (dd, 0.5H, J₁ = 7.8 Hz, J₂ = 3.4 Hz, Pro α-CH), 4.40 and 4.39 (s, 1H, 3′-CH), 4.32 (dd, 0.5H, J₁ = 8.5 Hz, J₂ = 4.0 Hz, Pro α-CH), 3.89–3.85 (m, 0.5H, Pro δ-CH₂), 3.78–3.67 (m, 0.5H, Pro δ-CH₂), 3.71 (s, 3H, OCH₃), 3.60–3.49 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.46–3.33 (m, 1H, 5-CH₂), 2.74–2.66 (m, 1H, 7′-CH₂), 2.29–1.84 (m, 9H, 8′-CH₂, Pro β-CH₂, Pro γ-CH₂, 3-CH₂, 4-CH₂), 1.83–1.76 (m, 2H, 4-CH₂, 7′-CH₂), 1.65 and 1.64 (s, 3H, 2′-C(CH₃)₂), 1.43 and 1.41 (s, 9H, C(CH₃)₃), 1.36 and 1.35 (s, 3H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 171.3 and 170.8 (lactam C=O), 170.7 and 170.6 (amide C=O), 170.1 (ester C=O), 154.6 and 153.7 (carbamate C=O), 79.5 and 79.4 (C(CH₃)₃), 71.9 and 71.8 (3′-C), 66.1 and 66.0 (6′-C), 62.0 and 61.9 (8′a-C), 58.1 and 58.0 (Pro α-C), 52.0 (OCH₃), 51.3 and 51.2 (2′-C), 48.4 and 48.3 (Pro δ-C), 47.2 and 46.9 (5-C), 37.7 and 37.6 (8′-C), 32.3 and 32.2 (2′-C(CH₃)₂), 31.8 and 31.5 (7′-C), 29.7 and 28.7 (3-C), 28.6 and 28.5 (C(CH₃)₃), 27.4 and 27.3 (Pro β-C), 24.6 and 24.5 (2′-C(CH₃)₂), 24.5, 24.3, 24.2, and 23.8 (Pro γ-C, 4-C); ESI HRMS m/z 518.2312 [M + Na]⁺, (C₂₄H₃₈N₃O₆S + Na⁺) requires 518.2295.

Methyl [3′R-(3′β,6′α,8′aβ)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (9b)

The same procedure used above to make 9a was used to convert spiro bicyclic compound 7b (115 mg, 0.3 mmol) to 110 mg (77%) of 9b as a clear oil: TLC Rf 0.55 (CH₂Cl₂/MeOH, 20:1); [α]_D −132 (c 3.02, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.03 (dt, 1H, 8′a-CH), 4.69 and 4.67 (s, 1H, 3′-CH), 4.38 (dd, 0.5H, J₁ = 7.9 Hz, J₂ = 3.5 Hz, Pro α-CH), 4.30 (dd, 0.5H, J₁ = 8.4 Hz, J₂ = 3.6 Hz, Pro α-CH), 3.85–3.79 (m, 0.5H, Pro δ-CH₂), 3.68 and 3.67 (s, 3H, OCH₃), 3.68–3.64 (m, 0.5H, Pro δ-CH₂), 3.58–3.46 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.41–3.28 (m, 1H, 5-CH₂), 2.56–2.42 (m, 2H, 8′-CH₂, 7′-CH₂), 2.28–1.82 (m, 9H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.79–1.69 (m, 1H, 4-CH₂), 1.56 and 1.55 (s, 3H, 2′-C(CH₃)₂), 1.39–1.34 (m, 12H, 2′-C(CH₃)₂, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC) δ 171.2 and 170.9 (lactam C=O), 170.5 and 170.2 (amide C=O), 169.13 and 169.11 (ester C=O), 154.6 and 153.7 (carbamate C=O), 79.3 and 79.2 (C(CH₃)₃), 71.9 and 71.8 (3′-C), 64.9 and 64.8 (6′-C), 62.9 and 62.8 (8′a-C), 58.1 and 58.0 (Pro α-C), 52.1 and 52.0 (OCH₃), 50.9 and 50.7 (2′-C), 48.1 and 48.0 (Pro δ-C), 47.1 and 46.8 (5-C), 41.5 and 41.4 (3-C), 36.5 and 36.3 (8′-C), 29.7 (Pro β-C), 28.9, 28.8, 28.7, and 28.6 (7′-C, 2′-C(CH₃)₂), 28.5 (C(CH₃)₃), 26.8 and 26.7 (2′-C(CH₃)₂), 24.4, 23.69, 23.68, 23.6 (Pro γ-C, 4-C); ESI HRMS m/z 518.2298 [M + Na]⁺, (C₂₄H₃₇N₃O₆S + Na⁺) requires 518.2295.

Methyl [3′S-(3′α,6′α,8′aα)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (10a)

The same procedure used above to make 9a was used to convert spiro bicyclic compound 8a (380 mg, 0.9 mmol) to 380 mg (81%) of 10a as white solid: mp 140–142 °C; TLC Rf 0.35 (CH₂Cl₂/MeOH, 20:1); [α]_D +57.9 (c 0.63, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.33 (dt, 1H, 8′a-CH), 4.52 (s, 1H, 3′-CH), 4.47 (dd, 0.5H, J₁ = 7.5 Hz, J₂ = 3.4 Hz, Pro α-CH), 4.33 (dd, 0.5H, J₁ = 8.0 Hz, J₂ = 3.2 Hz, Pro α-CH), 3.88 (dt, 0.5H, J₁ = 7.7 Hz, J₂ = 3.1 Hz, Pro δ-CH₂), 3.74 (dt, 0.5H, J₁ = 8.9 Hz, J₂ = 3.1 Hz, Pro δ- CH₂), 3.69 (s, 3H, OCH₃), 3.61–3.50 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.41–3.29 (m, 1H, 5-CH₂), 2.88–2.74 (m, 1H, 7′-CH₂), 2.27–1.69 (m, 11H, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 3-CH₂, 4-CH₂), 1.57 and 1.56 (s, 3H, 2′-C(CH₃)₂), 1.45–1.44 (m, 3H, 2′-C(CH₃)₂), 1.42 and 1.39 (s, 9H, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC) δ 171.0 and 170.6 (lactam C=O), 170.5 and 170.3 (amide C=O), 169.1 and 169.0 (ester C=O), 154.6 and 153.7 (carbamate C=O), 79.4 and 79.3 (C(CH₃)₃), 72.5 and 72.4 (3′-C), 66.1 and 66.0 (6′-C), 62.4 and 62.3 (8′a-C), 58.0 and 57.9 (Pro α-C), 51.9 and 51.8 (OCH₃), 51.4 and 51.3 (2′-C), 48.4 and 48.3 (Pro δ-C), 47.0 and 46.8 (5-C), 37.94 and 37.93 (8′-C), 31.0 and 30.8 (3-C), 30.3 and 30.2 (2′-C(CH₃)₂), 29.7 (Pro β-C), 28.7, 28.6, 28.5, and 28.3 (7′-C, C(CH₃)₃), 27.13 and 27.10 (2′-C(CH₃)₂), 24.3, 24.1, and 23.5 (Pro γ-C, 4-C); ESI HRMS m/z 518.2293 [M + Na]⁺ (C₂₄H₃₈N₃O₆S + Na⁺) requires 518.2295.

Methyl [3′S-(3′α,6′α,8′aβ)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxylate (10b)

The same procedure used above to make 9a was used to convert spiro bicyclic compound 8b (380 mg, 0.9 mmol) to 400 mg (84%) of 10b as a white solid: mp 162–163 °C; TLC Rf 0.40 (CH₂Cl₂/MeOH, 20:1); [α]_D −125 (c 0.95, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 4.96–4.91 (m, 1H, 8′a-CH), 4.45 (app. t, 0.5H, Pro α-CH), 4.32 (dd, 0.5H, J₁ = 7.9 Hz, J₂ = 3.0 Hz, Pro α-CH), 4.21 and 4.20 (s, 1H, 3′-CH), 3.87–3.81 (m, 0.5H, Pro δ-CH₂), 3.72–3.65 (ovlp. m, 0.5H, Pro δ-CH₂), 3.683 and 3.681 (s, 3H, OCH₃), 3.62–3.52 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.41–3.29 (m, 1H, 5-CH₂), 2.67–2.57 (m, 2H, 8′-CH₂, 7′-CH₂), 2.19–1.73 (m, 10H, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 3-CH₂, 4-CH₂), 1.59 and 1.58 (s, 3H, 2′-C(CH₃)₂), 1.43–1.40 (s, 9H, C(CH₃)₃), 1.37 and 1.36 (s, 3H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 171.7 and 170.4 (lactam C=O), 171.0 and 170.8 (amide C=O), 169.8 and 169.7 (ester C=O), 154.7 and 153.8 (carbamate C=O), 79.4 and 79.2 (C(CH₃)₃), 73.7 and 73.6 (3′-C), 64.9 and 64.8 (6′-C), 62.6 and 62.5 (8′a-C), 58.2 (Pro α-C), 52.1 and 51.9 (2′-C), 51.04 and 51.01 (OCH₃), 47.9 (Pro δ-C), 47.1 and 46.9 (5-C), 42.2 and 42.1 (3-C), 37.2 and 37.1 (8′-C), 32.2 and 31.1 (2′-C(CH₃)₂), 29.7 (Pro β-C), 28.9 and 28.7 (7′-C), 28.6 and 28.5 (C(CH₃)₃), 24.2, 23.8, and 23.6 (Pro γ-C, 2′-C(CH₃)₂), 23.6 and 23.5 (4-C); ESI HRMS m/z 518.2293 [M + Na]⁺, (C₂₄H₃₈N₃O₆S + Na⁺) requires 518.2295.

[3′R-(3′β,6′α,8′aβ)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (11)

The spirobicyclic methyl ester 9b (430 mg, 0.86 mmol) was dissolved in 9 mL of THF. LiOH (81.6 mg, 3.4 mmol) dissolved in 3 mL of H₂O was added to the above solution, and the reaction was stirred under Ar for 17 h at which time analysis by TLC indicated complete consumption of the starting material. THF was removed by rotary evaporation. The remaining aqueous residue was diluted with water while the pH was adjusted to 12–14 with 1 N NaOH. The solution was washed with 50 mL of EtOAc. The pH of the aqueous layer was then adjusted to 2–3 with dilute HCl, and the solution washed with EtOAc (2 × 50 mL). The organic layer was dried over MgSO₄, filtered, and concentrated to give a colorless oil which converted to a foamlike solid under high vacuum. The pure carboxylic acid weighed 413 mg (quantitative yield). ¹H NMR analysis indicated that the product was a single diastereoisomer and that the ester hydrolysis had taken place without any epimerization/racemization of any stereo centers in the molecule. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (br. s, 1H, COOH), 5.06 (dt, J₁ = 12.6 Hz, J₂ = 2.9 Hz, 1H, 8′a-CH), 4.69 and 4.66 (s, 1H, 3′-CH), 4.41 (dd, J₁ = 7.7 Hz, J₂ = 3.5 Hz, 0.5H, Pro α-C), 4.32 (dd, J₁ = 8.3 Hz, J₂ = 4 Hz, 0.5H, Pro α-CH), 3.88–3.83 (m, 0.5H, Pro δ-CH₂), 3.71–3.66 (m, 0.5H, Pro δ-CH₂), 3.61–3.30 (m, 3H, Pro δ-CH₂, 5-CH₂), 2.60–2.40 (m, 2H, 8′-CH₂, 7′-CH₂), 2.30–1.85 (m, 9H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.80–1.70 (m, 1H, 4-CH₂), 1.60 and 1.59 (s, 3H, 2′-C(CH₃)₂), 1.47 and 1.46 (s, 3H, 2′-C(CH₃)₂), 1.40 and 1.38 (m, 9H, C(CH₃)₃).

A solution of the carboxylic acid (413 mg, 0.86 mmol) in 3 mL of DMF was cooled to 0 °C. This was followed by the sequential addition of DIPEA (0.3 mL, 1.72 mmol) and HATU (392 mg, 1.03 mmol). The reaction was stirred for 20 min at this temperature under Ar. The solution was then cooled to −78 °C. In a cooled measuring cylinder, 2–3 mL of anhydrous ammonia was condensed into 5 mL of anhydrous CH₂Cl₂. This solution was added to the previous solution of the active ester. A canary yellow-colored solution was formed instantly. A glass adapter fitted with a balloon was attached to the reaction flask. The reaction was stirred at −78 °C for 7 h and then warmed to rt. The next day, another 2 mL portion of liquid ammonia was added and the reaction stirred for another 17 h. At the end of this time, the reaction mixture was diluted with 50 mL of CH₂Cl₂ and filtered to remove the yellow-colored precipitate. The almost colorless filtrate was concentrated and washed sequentially with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated. The residue was purified by flash chromatography on a Combiflash Retrieve system with a prepacked Luknova 40 g SiO₂ cartridge employing a gradient elution of 0–15% MeOH/CH₂Cl₂ at a flow rate of 25 mL/min to give a cloudy colorless oil. The precipitated tetramethyl urea (TMU) was removed by filtering the product through a 0.45 μm membrane filter disk to give 80 mg (20% yield) of pure 11. TLC Rf 0.35 (CH₂Cl₂/MeOH, 10:1); [α]_D −162.4 (c 1.15, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 6.28 (app. br t, 1H, NH), 5.95 (app. br d, 1H, NH), 5.11 (dt, 1H, 8′a-CH), 4.56 and 4.54 (s, 1H, 3′-CH), 4.41 (m, 0.5H, Pro α-CH), 4.30 (m, 0.5H, Pro α-CH), 3.86–3.80 (m, 0.5H, Pro δ-CH₂), 3.71–3.61 (m, 0.5H, Pro δ-CH₂), 3.55–3.45 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.43–3.30 (m, 1H, 5-CH₂), 2.55–2.40 (m, 2H, 8′-CH₂, 7′-CH₂), 2.30–1.85 (m, 9H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.77–1.70 (m, 1H, 4-CH₂), 1.57, 1.47 (s, 3H, 2′-C(CH₃)₂), 1.40 and 1.37 (m, 9H, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 171.3 and 170.9 (lactam C=O), 170.9, 170.5, 170.2 (amide C=O), 154.6 and 153.8 (carbamate C=O), 79.5 and 79.3 (C(CH₃)₃), 72.9 and 72.8 (3′-C), 65.0 and 64.9 (6′-C), 63.3 and 63.1 (8′a-C), 58.2 and 58.1 (Pro α-C), 50.8 and 50.6 (2′-C), 48.1 and 48.0 (Pro δ-C), 47.1 and 46.9 (5-C), 41.3 and 41.2 (3-C), 36.3 and 36.1 (8′-C), 29.7 (Pro β-C), 29.1, 29.0, 28.8, 28.7, 28.5, and 28.4 (2′-C(CH₃)₂, 7′-C, C(CH₃)₃), 26.5 (2′-C(CH₃)₂), 24.5, 23.9, 23.7 (Pro γ-C, 4-C); ESI HRMS m/z 503.2317 [M + Na]⁺, (C₂₃H₃₆N₄O₅S + Na⁺) requires 503.2299.

[3′R-(3′β,6′α,8′aβ)]-1-[[2″(S)-Pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide Trifluoroacetate Salt (4b·CF₃CO₂H)

The spiro-bicyclic carboxamide 11 (50 mg, 0.1 mmol) was dissolved in 2 mL of anhydrous CH₂Cl₂, and the solution cooled to 0 °C. Trifluoroacetic acid (0.4 mL, 5.1 mmol) was added dropwise to this solution which was then stirred at this temperature for 1 h and then allowed to warm to room temperature until the reaction was complete. The pale yellow-colored solution was concentrated to give an oil which was repeatedly triturated with a CH₂Cl₂/diethyl ether mixture and concentrated until a crystalline white-colored solid was obtained. The solid was dried well under high vacuum to give the final compound 4b weighing 49 mg (quantitative yield). A solution of the material (1.6 mg/mL in 4:1 acetonitrile–water) was diluted (2× by volume) with 0.05% ammonium formate buffer solution and 10–20 μL was subjected to HPLC analysis at 214 nm on a Varian Pursuit C18 column (3 μm particle size, 100 mm × 2 mm dimension) using a gradient of 10–90% acetonitrile in 0.05% ammonium formate buffer solution. A retention time of 4.24 min was obtained in a 10 min run, and purity was 96%. mp 260 °C (dec); TLC Rf 0.75 (iPrOH/NH₄OH, 4:1); [α]_D −163 (c 0.5, CH₃OH); ¹H NMR (400 MHz, CD₃OD, gCOSY assignment) δ 5.23 (dd, J₁ = 10 Hz, J₂ = 3.6 Hz, 1H, 8′a-CH), 4.52 (s, 1H, 3′-CH), 4.52–4.66 (m, 1H, Pro α-CH), 3.76–3.71 (m, 1H, Pro δ-CH₂), 3.57–3.51 (m, 1H, Pro δ-CH₂), 3.40–3.25 (m, 2H, 5-CH₂), 2.50–2.34 (m, 3H, 8′-CH₂, 7′-CH₂, 3-CH₂), 2.26–1.99 (m, 9H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.59, 1.50 (s, 3H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD, gHMQC and HMBC assignment) δ 170.6, 169.8, 165.8 (amide C=O), 74.0 (3′-C), 66.7 (6′-C), 63.9 (8′a-C), 60.4 (Pro α-C), 51.6 (2′-C), 48.7 (Pro δ-C, overlapped by residual solvent peaks, gHMQC assignment), 47.7 (5-C), 41.9 (3-C), 36.5 (Pro β-C), 29.7 (2′-C(CH₃)₂), 29.3 (8′-C), 29.2 (7′-C), 27.4 (2′-C(CH₃)₂), 25.5, 25.2 (Pro γ-C, 4-C); ESI HRMS m/z 381.1961 [M + H]⁺, (C₁₈H₂₉N₄O₃S + H⁺) requires 381.1955.

[3′S-(3′α,6′α,8′aα)]-1-[[1″-(tert-Butyloxycarbonyl)-2″(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (12)

The spirobicyclic methyl ester 10a (370 mg, 0.75 mmol) was hydrolyzed to its carboxylic acid in the same manner as described above for 9b in the experimental procedure for 11. The carboxylic acid was obtained in a quantitative yield (360 mg). ¹H NMR analysis indicated that the carboxylic acid was a single diastereoisomer. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (br. s, 1H, COOH), 5.23 (dt, J₁ = 10.4 Hz, J₂ = 3.2 Hz, 1H, 8′a-CH), 4.52 (s, 1H, 3′-CH), 4.41 (dd, J₁ = 7.6 Hz, J₂ = 2.8 Hz, 0.5H, Pro α-C), 4.31 (dd, J₁ = 8.0 Hz, J₂ = 3.6 Hz, 0.5H, Pro α-CH), 3.96 (t, J = 8.4 Hz, 0.5H, Pro δ-CH₂), 3.80 (t, J = 10.4 Hz, 0.5H, Pro δ-CH₂), 3.60–3.30 (m, 3H, Pro δ-CH₂, 5-CH₂), 2.51–1.67 (m, 12H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.80–1.70 (m, 1H, 4- CH₂), 1.64 (s, 3H, 2′-C(CH₃)₂), 1.54 (s, 3H, 2′-C(CH₃)₂), 1.41 and 1.39 (m, 9H, C(CH₃)₃).

The conversion of the carboxylic acid (262 mg, 0.54 mmol) to 12 was carried out in the same manner as described above for the synthesis of 11. The product was purified by flash chromatography on a Combiflash Retrieve system with a prepacked Luknova 40 g SiO₂ cartridge employing a gradient elution of 0–6% MeOH/CH₂Cl₂ at a flow rate of 30 mL/min. The pure product was obtained as a colorless oil weighing 130 mg (50% yield). TLC Rf 0.55 (CH₂Cl₂/MeOH, 10:1); [α]_D +59.3 (c 0.5, CHCl₃). ¹H and ¹³C NMR show the presence of two rotamers about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 7.39 and 7.32 (br s, 1H, NH), 5.15–5.11 (m, 2H, 8′a-CH, NH), 4.43 (dd, J₁ = 8.8 Hz, J₂ = 2.8 Hz, 0.5H, Pro α-CH), 4.36 (s, 1H, 3′-CH), 4.31 (dd, J₁ = 8.0 Hz, J₂ = 2.4 Hz, 0.5H, Pro α-CH), 3.95 (t, 0.5H, Pro δ-CH₂), 3.79 (t, 0.5H, Pro δ-CH₂), 3.60–3.31 (m, 3H, Pro δ-CH₂, 5-CH₂), 2.59–2.47 (m, 1H, 3-CH₂), 2.36–2.22 (m, 2H, 8′-CH₂, 4-CH₂), 2.15–1.72 (m, 9H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro β-CH₂, Pro γ-CH₂, 4-CH₂), 1.66 (s, 3H, 2′-C(CH₃)₂), 1.54 (s, 3H, 2′-C(CH₃)₂), 1.41 and 1.39 (m, 9H, C(CH₃)₃);¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 170.9, 170.5, 169.7, 169.5, 169.0, 168.8 (amide C=O), 153.3 and 152.3 (carbamate C=O), 79.7 and 79.6 (C(CH₃)₃), 73.3 (3′-C), 66.6 and 66.6 (6′-C), 62.4 and 62.3 (8′a-C), 58.2 (Pro α-C), 52.6 and 52.5 (2′-C), 48.9 (Pro δ-C), 47.5 and 47.2 (5-C), 37.3 (3-C), 31.3 and 31.1 (7′-C), 30.3 (Pro β-C), 29.6 and 29.5 (2′-C(CH₃)₂), 29.3 and 29.2 (8′-C, C(CH₃)₃), 27.7 and 27.6 (2′-C(CH₃)₂), 25.3, 25.0, 24.9, 24.5 (Pro γ-C, 4-C); ESI HRMS m/z 503.2317 [M + Na]⁺, (C₂₃H₃₆N₄O₅S + Na⁺) requires 503.2299.

[3′S-(3′α,6′α,8′aα)]-1-[[2″(S)-Pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide Trifluoroacetate Salt (3a·CF₃CO₂H)

The spiro-bicyclic carboxamide 12 (84 mg, 0.17 mmol) was deprotected with the same procedure as described above for the formaton of 4b. A white-colored solid was obtained in a yield of 71 mg (85%). A solution of this material (1.6 mg/mL in 4:1 acetonitrile–water) was diluted (2× by volume) with 0.05% ammonium formate buffer solution and 10–20 μL was subjected to HPLC analysis at 214 nm on a Varian Pursuit C18 column (3 μm particle size, 100 mm × 2 mm dimension) using a gradient 10–90% acetonitrile in 0.05% ammonium formate buffer solution. A retention time of 5.29 min was obtained in a 10 min run and purity was 99%. mp 260 °C (dec); TLC Rf 0.68 (iPrOH/NH₄OH, 5:1); [α]_D +80.4 (c 0.45, CH₃OH); ¹H NMR (400 MHz, CD₃OD, gCOSY assignment) δ 5.20 (dd, J₁ = 10.8 Hz, J₂ = 4.0 Hz, 1H, 8′a-CH), 4.87 (dd, J₁ = 8.8 Hz, J₂ = 5.2 Hz, 1H, Pro α-CH), 4.27 (s, 1H, 3′-CH), 3.76–3.72 (m, 1H, 5-CH₂), 3.53–3.47 (m, 1H, 5-CH₂), 3.33–3.22 (m, 2H, Pro δ-CH₂), 2.45–2.25 (m, 3H, 7′-CH₂, Pro β-CH₂, 8′-CH₂), 2.20–1.86 (m, 8H, 3-CH₂, 4-CH₂, Pro β-CH₂, Pro γ-CH₂, 7′-CH₂), 1.57, 1.49 (s, 3H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD, gHMQC and HMBC assignment) δ 171.1, 170.1, 166.3 (amide C=O), 74.4 (3′-C), 68.3 (6′-C), 63.4 (8′a-C), 60.3 (Pro α-C), 52.4 (2′-C), 49.5 (5-C, overlapped by residual solvent peaks, gHMQC assignment), 47.7 (Pro δ-C), 37.9 (3-C), 31.1 (7′-C), 29.9 (2′-C(CH₃)₂), 29.4 (Pro β-C), 28.1 (8′-C), 27.3 (2′-C(CH₃)₂), 25.5, 25.3 (Pro γ-C, 4-C); ESI HRMS m/z 381.1946 [M + H]⁺, (C₁₈H₂₉N₄O₃S + H⁺) requires 381.1955.

[3′S-(3′α,6′α,8′aα)]-1-(tert-Butyloxycarbonyl)-2,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (15a) and [3′S-(3′α,6′α,8′β)]-1-(tert-Butyloxycarbonyl)-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (15b)

N-Boc-α-butenylproline (5, 1.65 g, 6.1 mmol) was dissolved in a 60 mL mixture of EtOH and H₂O (1:1). The solution was then cooled in an ice/salt bath. Sodium bicarbonate (504 mg, 6.0 mmol) was added to the solution followed by d-penicillaminecarboxamide·HCl (13a, 1.1 g, 6.0 mmol). The pH of the resulting solution was adjusted to 6.5 and the colorless solution was stirred for 17 h at room temperature under Ar. After this time period, EtOH was removed in vacuo and the resulting aqueous residue was washed with EtOAc (3 × 100 mL). The pH of the aqueous layer was adjusted to 4.0–4.5 between the washes to ensure maximum product precipitation. The organic layers were combined and dried over MgSO₄, filtered, and concentrated to give the intermediate thiazolidine mixture of 14a as a white colored foam weighing 2.2 g (91% yield). The diastereoisomeric thiazolidine mixture (2.2 g, 5.5 mmol) was dissolved in 165 mL of anhydrous CH₂Cl₂. Triethylamine (1.9 mL, 13.7 mmol) was added in one portion to the solution followed by CMPI (1.7 g, 6.6 mmol). A bright yellow-colored suspension was obtained. This suspension was refluxed for 17 h at the end of which an amber-colored solution was obtained. The reaction mixture was washed sequentially with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange-colored syrup that was purified using flash chromatography on a Luknova 80 g SiO₂ gel prepacked column with a gradient elution of 40–100% EtOAc/hexanes followed by 1–5% MeOH/EtOAc. The two diastereoisomers, 15a and 15b, were obtained in a 2:5 ratio in a 71% yield.

15a

White foam (425 mg); mp 68–78 °C; TLC Rf 0.61 (MeOH/EtOAc, 2.5%); [α]_D +145.1 (c 1.11, CHCl₃); ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 7.47 (br.s, 1H, NH₂), 5.16 (dd, 1H, J₁ = 10.8 Hz, J₂ = 3.6 Hz, 8′a-CH), 5.07 (br. s, 1H, NH₂), 4.44 (s, 1H, 3′-CH), 3.55–3.44 (m, 2H, 5-CH₂), 2.45 (app. t, 1H, 7′-CH₂), 2.38–2.24 (m, 2H, 3-CH₂, 8′-CH₂), 2.02–1.74 (m, 5H, 4-CH₂, 3-CH₂, 7′-CH₂, 8′-CH₂), 1.63 (2′-C(CH₃)₂), 1.52 (2′-C(CH₃)₂), 1.33 (C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC assignment) δ 169.9, 169.8 (lactam and carboxamide C=O), 153.4 (carbamate C=O), 80.4 (C(CH₃)₃), 73.4 (3′-C), 65.2 (6′-C), 62.6 (8′a-C), 52.6 (2′-C(CH₃)₂), 48.9 (5-C), 38.4 (3-C), 32.2 (7′-C), 29.6 (2′-C(CH₃)₂), 29.4 (C(CH₃)₃), 27.8 (8′-C), 27.6 (2′-C(CH₃)₂), 24.4 (4-C); ESI HRMS m/z 406.1789 [M + Na]⁺, (C₁₈H₂₉N₃O₄S + Na⁺), requires 406.1771.

15b

White solid (1.1 g); suitable crystals for an X-ray structure¹⁵ were obtained after crystallization from hexanes, EtOAc, and isopropyl alcohol; mp 238–241 °C; TLC Rf 0.38 (MeOH/EtOAc, 2.5%); [α]_D – 43.9 (c 1.09, CDCl₃); ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 7.18 (br s, 1H, NH₂), 5.09 (br s, 1H, NH₂), 4.92 (dd, 1H, J₁ = 11.2 Hz, J₂ = 2.4 Hz, 8a′-CH), 4.15 (s, 1H, 3′-CH), 3.57–3.51 (m, 1H, 5-CH₂), 3.45–3.39 (m, 1H, 5-CH₂), 2.77 (dddd or dq, 1H, J₁ = J₂ = J₃ = 15.6 Hz, J₄ = 4 Hz, 8′-CH₂), 2.45–2.33 (m, 2H, 7′-CH₂, 3-CH₂), 2.11–1.94 (m, 3H, 8′-CH₂, 7′-CH₂, 4-CH₂), 1.86–1.76 (m, 2H, 4-CH₂, 3-CH₂), 1.60 (s, 3H, 2′-C(CH₃)₂), 1.48 (s, 3H, 2′-C(CH₃)₂), 1.37 (s, 9H, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 170.0, 169.2 (lactam and carboxamide C=O), 153.9 (carbamate C=O), 80.1 (C(CH₃)₃), 74.3 (3′-C), 62.9 (6′-C), 62.7 (8a′-C), 51.4 (2′-C(CH₃)₂), 48.9 (5-C), 42.9 (3-C), 38.9 (7′-C), 32.7 (2′-C(CH₃)₂), 29.3 (C(CH₃)₃), 27.8 (8′-C), 25.1 (2′-C(CH₃)₂), 24.7 (4-C); ESI HRMS m/z 406.1781 [M + Na]⁺, (C₁₈H₂₉N₃O₄S + Na⁺) requires 406.1771.

[3′R-(3′β,6′α,8′aα)]-1-[[1″-(tert-Butyloxycarbonyl)-2″-(S)-pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (18)

N-Boc-α-butenylproline (5, 542 mg, 2 mmol) was dissolved in a 20 mL mixture of EtOH and H₂O (1:1). The solution was then cooled in an ice/salt bath. Sodium bicarbonate (168 mg, 2 mmol) was then added to the solution followed by l-penicillaminecarboxamide·HCl (13b, 386 mg, 2.1 mmol). The pH of the resulting solution was adjusted to 6.5, and the colorless solution was stirred for 17 h at room temperature under Ar. After this time period, EtOH was removed in vacuo and the resulting aqueous residue was washed with EtOAc (3 × 75 mL). The pH of the aqueous layer was adjusted to 4.0–4.5 between the washes to ensure maximum product precipitation. The organic layers were combined and dried over MgSO₄, filtered, and concentrated to give the intermediate thiazolidine mixture of 14b as a white colored foam weighing 682 mg (85% yield).

The diastereomeric thiazolidine mixture (682 mg, 1.69 mmol) was dissolved in 50 mL of anhydrous CH₂Cl₂. Triethylamine (0.592 mL, 4.25 mmol) was added in one portion to the solution followed by CMPI (516 mg, 2.02 mmol, 1.2 equiv). A bright yellow-colored suspension was obtained. This suspension was refluxed for 17 h at the end of which an amber-colored solution was obtained. The reaction mixture was washed sequentially with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange-colored syrup. The mixture consisted of two diastereoisomers (17) that were obtained in a 1:2 ratio. These diastereoisomers were not separable on TLC, so the diastereoisomers were taken on the next reaction.

A solution of the diastereoisomeric spirocycle 17 (375 mg, 0.98 mmol) in 3 mL of anhydrous CH₂Cl₂ was cooled to 0 °C with an ice bath. Trifluoroacetic acid (3.7 mL, 49 mmol) was then added dropwise to this solution, which was then stirred at 0 °C until removal of the Boc-group was determined to be complete by TLC. This was usually between 1.5 and 2 h. The reaction was warmed to rt, and the volatiles removed by rotary evaporation. The residue was redissolved in CH₂Cl₂ and the solvent removed by evaporation twice until the excess TFA was removed. The pale yellow colored residue was dried to a constant weight for 5–6 h under vacuum.

A solution of Boc-l-proline (251 mg, 1.17 mmol) in 4 mL of DMF was cooled to −5 °C using an ice/salt bath. DIPEA (0.39 mL, 2.94 mmol) was added dropwise to this cooled solution followed by HATU (445 mg, 1.17 mmol). The clear solution was stirred for 30 min after which a solution of the diastereoisomeric spirocycle free amine in 4 mL of DMF, obtained by neutralizing the TFA salt (365 mg, 0.98 mmol) with DIPEA (0.393 mL, 2.94 mmol), was added to the active ester. The yellow colored solution was warmed to rt, and then it was stirred under Ar for 36 h. At the end of this time, DMF and other volatiles were removed in vacuo. The residue was dissolved in 120 mL of EtOAc, and this solution was sequentially washed with 10% citric acid, sat. NaHCO₃, and brine solutions. The organic layer was dried over MgSO₄, filtered, and concentrated to give yellow-colored residue. The diastereoisomers were separated by silica gel flash chromatography with a gradient elution consisting of 40–70% acetone/ethyl acetate in discrete steps to give 11 and 18 in a 1:2 ratio.

18

NOESY studies were carried out to delineate the relative stereochemistry. Both the 3′-H and the 8′a-H protons were irradiated in separate experiments, and NOE enhancements were observed in each case. The experiments confirmed that the bridgehead proton was cis to the 3′-hydrogen of the thiazolidine portion of the molecule. ¹H and ¹³C NMR showed the presence of two rotamers about the carbamate bond in a 1:1 ratio. mp 244 °C; TLC Rf 0.45 (acetone/ethyl acetate, 4:1); [α]_D −74.9 (c 1.0, CH₃OH); ¹H NMR (400 MHz, CDCl₃, gCOSY assignment) δ 5.95 (d, 1H, J = 4.92, NH), 5.8 (br s, 1H, NH), 5.07–5.13 (m, 1H, 8′a-CH), 4.38 (m, 0.5H, Pro α-CH), 4.27 (m, 0.5H, Pro α-CH), 4.17 and 4.16 (s, 1H, 3′-CH), 3.86–3.80 (m, 0.5H, Pro 5-CH₂), 3.71–3.67 (m, 0.5H, Pro 5-CH₂), 3.53–3.44 (m, 2H, Pro δ-CH₂, 5-CH₂), 3.38–3.25 (m, 1H, δ-CH₂), 2.69–2.58 (m, 1H, 3-CH₂), 2.22–1.68 (m, 11H, 3-CH₂, 8′-CH₂, 7′-CH₂, Pro γ-CH₂, Pro β-CH₂, 4-CH₂), 1.57 (s, 3H, 2′-C(CH₃)₂), 1.39, 1.37, and 1.34 (s, 12H, 2′-C(CH₃)₂, C(CH₃)₃); ¹³C NMR (100 MHz, CDCl₃, gHMQC and HMBC assignment) δ 171.9 and 171.8 (lactam C=O), 171.2, 170.7, 170.4, 170.2 (amide C=O), 154.4 and 153.5 (carbamate C=O), 79.4 and 79.2 (C(CH₃)₃), 73.2 and 73.1 (3′-C), 65.9 and 65.8 (6′-C), 62.9 and 61.8 (8′a-C), 57.9 and 57.8 (Pro α-C), 51.2 and 51.2 (2′-C), 48.2 and 48.1 (5-C), 47.0 and 46.7 (Pro δ-C), 37.3 and 37.2 (8′-C), 32.0 and 31.9 (2′-C(CH₃)₂), 31.7, 31.5 (3′-C), 29.6, 29.3, 28.4, 28.3, 27.6 (Pro β-C, C(CH₃)₃, 7′-C), 24.2, 24.2, (2′-C(CH₃)₂), 23.9 (Pro γ-C), 23.5 (4-C); ESI HRMS m/z 503.2428 [M + Na]⁺, (C₂₃H₃₆N₄O₅S + Na⁺) requires 503.2299.

[3′R-(3′β,6′α,8′aα)]-1-[[2″(S)-Pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide Trifluoroacetate Salt (4a·CF₃CO₂H)

Spiro-bicyclic carboxamide 18 (66 mg, 0.1 mmol) was dissolved in 2 mL of anhydrous CH₂Cl₂, and the solution was cooled to 0 °C. Trifluoroacetic acid (0.4 mL, 5.1 mmol) was added dropwise to this solution, which was then stirred at this temperature for 1 h. The reaction was warmed to room temperature where it was stirred until the reaction was complete. The pale yellow-colored solution was concentrated to give a oil, which was repeatedly triturated with a CH₂Cl₂/diethyl ether mixture and concentrated until a white-colored solid was obtained. The solid was dried under high vacuum to give 4a (49 mg) in a quantitative yield. The purity of the product was confirmed by HPLC with a Varian Pursuit C18 column (3 μm particle size, 100 mm × 2 mm dimension) and acetonitrile/H₂O (98:2) as the solvent system. A retention time of 4.24 min was obtained in a 30 min run and purity was 95%. mp 246 °C; TLC Rf 0.71 (iPrOH/NH₄OH, 5:1); [α]_D −23.9 (c 1.0, CH₃OH); ¹H NMR (400 MHz, CD₃OD, gCOSY assignment) δ 5.17 (dd, J₁ = 3.0, J₂ = 3.04 Hz, 1H, 8′a-CH), 4.53 (d, J = 2.76 Hz, 1H, Pro α-CH), 4.20 (s, 1H, 3′-CH), 3.82–3.77 (m, 1H, 5-CH₂), 3.58–3.48 (m, 1H, 5-CH₂), 3.40–3.33 (m, 2H, Pro δ-CH₂), 2.52–2.46 (m, 2H, 3′-CH₂, 8′-CH₂), 2.23–1.94 (m, 10H, 3-CH₂, 8′-CH₂, 4-CH₂, Pro β-CH₂, Pro γ-CH₂, 7′-CH₂), 1.64, 1.44 (s, 6H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD, gHMQC and HMBC assignment) δ 173.5, 171.7, 167.3 (amide C=O), 74.1 (3′-C), 68.1(6′-C), 63.4 (8′a-C), 60.5 (Pro α-C), 51.8 (2′-C), 49.0 (5-overlapped by residual solvent peaks, gHMQC assignment C), 47.5 (Pro δ-C), 38.4 (7′C), 32.3 (3-C), 32.0 (2′-C(CH₃)₂), 28.7(8′-C), 27.3 (Pro β-C), 25.1, 25.0 (Pro γ-C, 4-C), 24.5 (2′-C(CH₃)₂; ESI HRMS m/z 381.1874 [M+H]⁺, (C₁₈H₂₉N₄O₃S + H⁺) requires 381.1955.

[3′S-(3′α,6′α,8′aβ)]-1-[[1″-(tert-Butyloxycarbonyl)-2″-(S)-pyrrolidinyl]carbonyl]- 2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide (16)

A solution of the spirocycle 15b (150 mg, 0.391 mmol) in 2 mL of anhydrous CH₂Cl₂ was converted to 16 by the same procedure used to convert 17 to 18. The product was purified by flash chromatography with a gradient elution consisting of 0–5% MeOH/CH₂Cl₂ in discrete steps to give a lightly pink-colored foam of 108 mg (58% yield). TLC Rf 0.58 (MeOH/CH₂Cl₂, 1:9); [α]_D 24.5 (c 1.0, CH₃OH). NMR spectra indicated the presence of two rotameric forms of the molecule about the carbamate bond in a 1:1 ratio. ¹H NMR (400 MHz, CDCl₃, gCOSY) δ 7.36 and 7.28 (s, 1H, carboxamide NH), 5.16 (d. 1H, J = 7.9 Hz, carboxamide NH), 5.09 (dd, 1H, J₁ = 3.64, 3.76, 8′a-CH), 4.38 (dd, 0.5H, J = 1.92, 2.52 Hz, Pro α-CH), 4.33 (s, 1H, 3′-CH), 4.28 (dd, 0.5H, J = 2.28, 3.08 Hz, Pro α-CH), 3.91 (t, 0.4H, J = 3.69, 3.92 Hz, Pro δ-CH), 3.74 (dd, 0.6H, J = 2.8, 8.48 Hz, Pro δ-CH), 3.56–3.27 (m, 3H, Pro δ-CH, 5-CH₂), 2.49 (m, 1H, 8′-CH), 2.33–1.79 (m, 11H, 7′-CH₂, 8′-CH₂, Pro β-CH₂, Pro γ-CH₂, 3-CH₂, 4-CH₂), 1.62 (s, 3H, 2′-C(CH₃)₂), 1.51 (s, 3H, 2′-C(CH₃)₂), 1.38 and 1.35 (s, 9H, Boc-CH₃); ¹³C NMR (100 MHz, CDCl₃, HMQC) δ 172.3, 171.9, 171.2, 172.0, 170.4, and 170.2 (amide C=O), 154.5 and 153.5 (Boc C=O), 79.6 and 79.5 (Boc C(CH₃)₃), 73.1 (3′-C), 66.39, 66.37 (8′a-C), 62.05, 62.01 (6′-C), 57.8 (Pro α-C), 52.10 and 51.99 (2′-C), 48.3 (Pro -δC), 46.9 and 46.6 (Pro −5C), 36.6 (3-C), 30.5 and 30.2 (8′-C), 29.4 (Pro β-C), 28.8, 28.6, 28.4, 28.3 (2′-C(CH₃)₂, Boc C(CH₃)₃, 26.8, 26.6 (2′-C(CH₃)₂), 24.6, 24.5, 23.5 (Pro γ-C and 4-C); ESI HRMS m/z 503.2333 [M + Na]⁺, (C₂₃H₃₆N₄O₅S + Na⁺) requires 503.2299.

[3′S-(3′α,6′α,8′β)]-1-[[2″(S)-Pyrrolidinyl]carbonyl]-2′,2′-dimethyl-5′-oxospiro[pyrrolidine-2,6′-thiazolidino[3,2-a]piperidine]-3′-carboxamide Trifluoroacetate Salt (3b)

The spiro bicyclic carboxamide 16 (84 mg, 0.17 mmol) was deprotected in the same manner as described above for the conversion of 18 to 4a. The product was obtained in a yield of 81 mg (97%). The purity of the product was confirmed by HPLC with a Varian Pursuit C18 column (3 μm particle size, 100 mm × 2 mm dimension) with acetonitrile/H₂O (98:2) as the solvent system. A retention time of 4.2 min was obtained in a 30 min run and the purity was 99%. mp 237 °C (dec); TLC Rf 0.74 (iPrOH/NH₄OH, 4:1); [α]_D −56.9 (c 0.49, CH₃OH); ¹H NMR (400 MHz, CD₃OD, gCOSY assignment) δ 5.15 (dd, J₁ = 2.64, J₂ = 2.28 Hz, 1H, 8′a-CH), 4.54 (dd, J₁ = 5.13, J₂ = 5.92 Hz, 1H, Pro α-CH), 4.11 (s, 1H, 3′-CH), 3.81–3.76 (m, 1H, 5-CH₂), 3.59–3.53 (m, 1H, 5-CH₂), 3.43–3.40 (m, 2H, Pro δ-CH₂), 2.63–2.41 (m, 3H, 7′-CH₂, 8′-CH₂), 2.20–1.86 (m, 10H, 3-CH₂, 4-CH₂, Pro γ-CH₂, Pro β-CH₂, 7′-CH₂), 1.65, 1.44 (s, 6H, 2′-C(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD, gHMQC and HMBC assignment) δ 173.6, 171.6, 168.0 (amide C=O), 75.3 (3′-C), 66.5 (6′-C), 63.0 (8′a-C), 60.5 (Pro α-C), 52.0(2′-C), 48.3 (5-C overlapped by residual solvent peaks, gHMQC assignment), 47.7 (Pro δ-C), 42.3 (7′-C), 37.7 (3-C), 32.0 (2′-C(CH₃)₂), 29.0 (Pro β-C), 28.6 (8′-C), 25.18, 25.12 (Pro γ-C, 4-C), 24.3 (2′-C(CH₃)₂); ESI HRMS m/z 381.1966 [M + H]⁺, (C₁₈H₂₉N₄O₃S + H⁺) requires 381.1955.

Dopamine D₂ Receptor Binding Assay

The effect of compounds 3a, 3b, 4a, and 4b on [³H]NPA binding in striatal membranes was analyzed using receptor binding studies. Bovine striatal membranes were prepared as previously described by Kazmi et al.,¹⁸ where freshly dissected calf bovine striatum was homogenized and centrifuged in buffer to isolate cell membranes. [³H]NPA was synthesized through a custom order from Vitrax Radiochemicals (Placentia, CA) and was provided with a specific activity of 60 Ci/mmol. Receptor modulatory assays were performed in a similar manner to Srivastava et al.,⁴ wherein 100 μg of striatal membrane were prepared and incubated with the compounds at concentrations ranging from 10^–5 to 10^–10 M in the presence of [³H]NPA. The reaction was incubated in a total 500 μL volume of assay buffer (50 mM Tris-HCl, 5 mM KCl, 4 mM MgCl₂, 1 mM ethylenediaminetetraacetic acid, and 120 mM NaCl (pH 7.4)). Reaction mixtures were incubated for 1 h at 25 °C and subsequently subjected to rapid filtration. Radioactivity-bound filter disks were placed in plastic scintillation vials containing 4 mL of scintillation fluid, equilibrated for at least 12 h, and counted in a Beckman LS5000 liquid scintillation counter (model LS5 KTA). Specific binding was defined as the difference between the radioactivity bound in the absence and presence of 10 μM sulpiride (Sigma).

Concentration response curves with varying concentrations of [³H]NPA were generated under the same conditions as described above for the modulatory assay. As described previously, 100 μg of striatal membranes were prepared and incubated with compounds 3a and 4a at effective concentrations of 1 and 10 μM, respectively. The reaction was incubated in a total 500 μL volume of assay buffer (pH 7.4) with concentrations of [³H]NPA ranging from 10^–9 to 10^–15 M. Reaction mixtures were incubated for 1 h at 25 °C and subsequently subjected to rapid filtration. Radioactivity-bound filter disks were placed in plastic scintillation vials containing 4 mL of scintillation fluid, equilibrated for at least 12 h, and counted in a Beckman LS5000 liquid scintillation counter. Specific binding was measured in the absence and presence of 10 μM sulpiride.

Supporting Information Available

Synthetic scheme and experimental details of the synthesis of d- and l-penicillamine carboxamides 13a and 13b. ORTEP drawing of the X-ray crystal structure of compounds 7b, 8a, 8b, 15b, and S2a. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

S.B. and S.M. carried out the synthesis, purification, and characterization of the allosteric modulators. R.D. and J.M. carried out the pharmacological evaluation of the allosteric modulators. R.K.M. directed the pharmacological work. R.L.J. directed the synthetic work and wrote the paper based upon drafts written by S.B. and S.M.

This work was supported by Grant Number NS20036 from NIH/NINDS to R.L.J. and by Grant Number S10RR024661 from the National Center for Research Resources.

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States