Synthesis and Antiproliferative Activity of Phosphorus Substituted 4-Cyanooxazolines, 2-Aminocyanooxazolines, 2-Iminocyanooxazolidines and 2-Aminocyanothiazolines by Rearrangement of Cyanoaziridines

Several phosphorus-substituted N-acylated cyanoaziridines 2 and N-carbamoylated cyanoziridines 5 were prepared in good to high yields. N-Acylated cyanoaziridines 2 were used, after ring expansion, in an efficient synthesis of oxazoline derivative 3a and in a completely regio-controlled reaction in the presence of NaI. Conversely, N-carbamoyl cyanoaziridines 5 reacted with NaI to obtain a regioisomeric mixture of 2-aminocyanooxazolines 7. Mild acidic conditions can be used for the isomerization of N-thiocarbamoyl cyanoaziridine 6a into a 2-aminocyanothiazoline derivative 8a by using BF3·OEt2 as a Lewis acid. Likewise, a one pot reaction of NH-cyanoaziridines 1 with isocyanates obtained 2-iminocyanooxazolidines 9 regioselectively. This synthetic methodology involves the addition of isocyanates to starting cyanoaziridines to obtain N-carbamoyl cyanoaziridines 5, which after the ring opening, reacts with a second equivalent of isocyanate to give the final 2-imino cyanooxazolidines 9. In addition, the cytotoxic effect on the cell lines derived from human lung adenocarcinoma (A549) was also screened. 2-Iminooxazolidines 9 exhibited moderate activity against the A549 cell line in vitro. Furthermore, a selectivity towards cancer cells (A549) over non-malignant cells (MCR-5) was detected.


Introduction
DNA-modifying agents are a significant class of pharmaceuticals used in conventional chemotherapy. Aziridine-based cytostatic compounds, acting as powerful alkylating agents, have an inherent in vivo potency due to their ability to act as DNA cross-linking agents via the ring opening of aziridine [1]. Mitomycin C and many variants of this natural product have been well characterized for their anti-tumor activity based on the nucleophilic ring opening of the three-membered nitrogen heterocycle, leading to the alkylation of DNA [2]. Mitomycin C is a conventional DNA cross-linking agent that uses the reductive activation of the aziridine moiety to form lethal DNA-DNA cross-links, as well as, more often, mono-alkylated DNA products [3]. Founded on this knowledge, during the early 1970s Bicker [4,5] developed a variety of 2-cyanoaziridine derivatives as potential carcinostatic agents. For instance, 2-cyanoaziridine-1-carboxamide (Ia, Figure 1) was active against a PIE 2-3 sarcoma in Wistar rats, and it had a low toxicity. However, it showed weak antitumor activity in cell cultures. The fact that it increased rather than decreased the number of leukocytes was especially interesting [5]. However, in contrast to the initial findings, these cyanoaziridines showed no alkylating activity in vitro or in vivo [4]. These results suggest that the cyano group reduces the reactivity required for the alkylation of DNA bases and that they may selectively react with sulfur moieties in Furthermore, the ring-expansion of aziridines with isocyanates has been revealed to be a useful pathway for the synthesis of a variety of five-membered nitrogen-containing compounds. For instance, KI [17], NaI [18][19][20], NiI 2 [21], Pd-catalyzed [22][23][24] reactions of aziridines with isocyanates, or even in the absence of catalysts [25], have been described. All these reactions yielded imidazolidin-2-ones or oxazolidin-2-imines compounds which generate great interest in medicinal and pharmaceutical chemistry since they represent classes of heterocyclic compounds with a broad variety of biological activities [26,27]. In the last few years we have been involved in the chemistry related to phosphorylated 2H-azirines for the preparation of αor β-aminophosphonic acid derivatives [28][29][30], pyrroles [31,32], oxazoles [33], 1H-benzo[d]azepines [31], or hybrid molecules such as azirino [2,1-b]benzo[e] [1,3]oxazines [34], among others. Additionally, organophosphorus derivatives are fascinating compounds from a biological point of view, due to the fact that these substituents may regulate relevant biological functions modifying the reactivity of heterocyclic systems [35]. Recently, we have revealed a diastereoselective method for the preparation of phosphorus substituted cyanoaziridines by means of the nucleophilic addition of TMSCN as a cyanide source to the C-N double bond of 2H-azirines [16]. Following our earlier studies on the preparation of phosphorylated cyanoaziridines, here we wish to report the synthesis of structurally new phosphorus substituted N-(thio)carbamoyl cyanoaziridines by the coupling of unactivated cyanoaziridines with iso(thio)cyanates. Moreover, the ring expansion into 2-aminocyanooxazolines, 2-aminocyanothiazolines, or 2-iminocyanooxazolidines under nucleophilic or acidic conditions is also explored. We also focus on their biological activity and highlight the antiproliferative effect of all these new heterocycles on A549 human lung adenocarcinoma cells.

Chemistry
As a continuation of the studies on the synthesis and applications of activated aziridines through the N-functionalization of unactivated cyanoaziridines [16], and taking into account that N-acylaziridines are very important synthons in the development of new ring opening aziridine reactions, we initially studied the N-acylation reaction of cyanoaziridine phosphine oxides 1a (R = Ph, R 1 = Me) and 1b (R = Ph, R 1 = Et), and phosphonate 1c (R = OEt, R 1 = Me). Thus, the N-acylation of cyanoaziridines 1a-c using 3,5-dinitrobenzoyl chloride in the presence of a base, such as Et 3 N, and methylene chloride as the solvent, obtained N-acylated cyanoziridines 2a-c in good chemical yields (80-93%) (Scheme 1).

Scheme 1. Synthesis of N-acylated cyanoaziridines phosphine oxides and phosphonates 2.
We next studied the isomerization reaction (Heine reaction) of the corresponding synthesized phosphorus-containing N-acylated cyanoaziridines 2. For this purpose, we chose nucleophilic conditions [36][37][38][39], and the isomerization of benzoyl aziridine 2a was accomplished by the use of the sodium iodide method. Indeed, when phosphorus substituted N-acylated cyanoaziridine 2a reacted with 0.2 equivalents of NaI in THF at 60 • C in a sealed-tube, 4-cyanooxazoline derived from phosphine oxide 3a was obtained in a regioselective way in a 65% yield (Scheme 2). Even though the iodide anion may attack both the aziridine carbon atoms, and the regioselectivity will be determined by the stereoelectronic nature of the substituents, several reports in the literature describe the aziridine ring opening through the less hindered position [40][41][42]. Bear in mind that the most reasonable mechanism may consider a first step where the iodide anion would attack at the C2 aziridine carbon that was substituted less in a regiospecific manner, followed by the aziridine ring opening obtaining intermediate 4.
The iodide displacement in the former intermediate may afford phosphorus substituted 4-cyanooxazoline 3a (Scheme 2).
Unactivated NH-aziridines are very stable compounds in basic conditions and may easily react with electrophiles. Next, we explored the N-functionalization of unactivated cyanoaziridines derived from phosphine oxides and phosphonates 1 with aromatic and aliphatic isocyanates for the preparation of functionalized N-aryl or N-alkylcarbamoyl cyanoaziridines.
Therefore, the reaction of cyanoaziridines 1 with isocyanates was assessed. Thus, as outlined in Table 1, in an initial experiment the addition of phenyl isocyanate to cyanoaziridine phosphine oxide 1a (R = Ph, R 1 = Me) was readily achieved in dichloromethane at room temperature. A total of 1.2 equivalents of the isocyanate component were used in order to ensure a full conversion. Under these reaction conditions, the corresponding phosphorus substituted N-phenylcarbamoyl cyanoaziridine 5a (R = R 2 = Ph, R 1 = Me) was obtained in a 63% yield (Table 1, entry 1). Some examples in the literature describe the use of Lewis acids as transition metal catalysts involving reactions of aziridines and isocyanates [22,43,44]. Hence, we explored the reaction of phenyl isocianate with cyanoaziridine 1a in the presence of a Lewis acid. Better yields and reduced reaction times were observed for the synthesis of 5a using catalytic ammounts of Sc (OTf) 3 (20%) (see Table 1, entry 2). In the same way, phosphatederived cyanoziridine 1c (R = OEt, R 1 = Me) reacted with phenyl isocyanate in CH 2 Cl 2 at room temperature, without a catalyst, to give N-functionalized cyanoaziridine 5b (R = OEt, R 1 = Me, R 2 = Ph) in very good chemical yields (Table 1, entry 3).
This process was extended to the reactivity of phosphorus substituted cyanoziridines 1 with alkyl isocyanates, such as ethyl and tert-butyl isocyanate. The same reaction conditions used for the aromatic isocyanates were employed for the reaction of 1a with ethyl isocyanate. Nevertheless, no progress was observed on the formation of compound 5g, and the starting cyanoaziridine 1a was recovered instead. The Lewis acid activation of the aziridine ring in the reaction of 1a with ethyl isocyanate led to the formation of the expected compound 5g. Thus, the presence of 20% mol of Sc (OTf) 3 as a Lewis acid, as described before for the synthesis of 5a, gave only a 50% conversion of N-ethylcarbamoyl cyanoaziridine 5g (R = Ph, R 1 = Me, R 2 = Et) after 24 h of the reaction. However, when two equivalents of ethyl isocyanate reacted with cyanoaziridine 1a in the presence of 1.25 equivalents of ZnCl 2 in CH 2 Cl 2 and at room temperature, N-functionalized cyanoaziridine 5g was obtained with a 86% chemical yield ( Table 1, entry 8). Similarly, the addition of ethyl isocyanate to phosphonate-derived cyanoaziridine 1c, using ZnCl 2 as a Lewis acid, led to the formation of N-functionalized aziridine 5h (R = OEt, R 1 = Me, R 2 = Et; Table 1, entry 9). In addition, the synthesis of N-tert-butylcarbamoyl cyanoaziridines 5i-k (Table 1, entries 10-12) was achieved in moderate yields, using tert-butyl isocyanate as an electrophile and ZnCl 2 as a Lewis acid.
The synthetic procedure for the preparation of N-aryl or N-alkylcarbamoyl cyanoaziridines 5 could be widened to the addition of isothiocyanates to cyanoaziridines 1 ( Table 2). Under the same reaction conditions used for the preparation of derivatives 5, phenyl isothiocyanate, pmethoxyphenyl isothiocyanate, or p-nitrophenyl isothiocyanate did not react with cyanoaziridine 1a to yield compounds 6. Moreover, the use of different bases, such as Et 3 N, pyridine, NaH, or Cs 2 CO 3 , as well as Lewis acids such as ZnCl 2 or Sc (OTf) 3 , gave similar results: the formation of N-functionalized cyanoaziridines 6 was not observed and the starting compound 1a was recovered instead. In order to achieve the synthesis of new derivatives 6, we decided to use a more reactive isothiocyanate derivative. Thus, functionalized isothiocyanates with an electronwithdrawing group, such as ethoxycarbonyl isothiocyanate, reacted with cyanoaziridines derived from phosphine oxide 1a and 1b in CH 2 Cl 2 at −30 • C (Method A). Under these reaction conditions, compounds 6a and 6b were attained in 71% and 85% chemical yields, respectively ( Table 2, entries 1 and 3). Increasing the reaction temperature to 25 • C (Method B) gave better yields and the N-functionalized cyanoaziridine 6a was obtained in an 80% yield ( Table 2, entry 2). Similarly, phosphonate-derived cyanoaziridine 1c reacted with ethoxycarbonyl isothiocyanate in CH 2 Cl 2 at room temperature to afford N-thiocarbamoyl cyanoaziridine 6c (R = OEt, R 1 = Me) in good yields ( Table 2, entry 4).
Continuing with our interest in the synthesis of new 5-membered nitrogen containing heterocyclic compounds, we then explored the ring expansion of some N-carbamoyl cyanoaziridine derivatives 5. To this end, and using the same reaction conditions as in the case of N-acyl cyanoaziridine 2, N-arylcarbamoyl cyanoaziridines derived from phosphine oxide 5a (R = R 2 = Ph) and 5c (R = Ph, R 2 = p-MeC 6 H 4 ) reacted with 0.2 equivalents of NaI at 60 • C in THF, allowing the preparation of oxazolines 7a and 7c, respectively (Table 3, entries 1 and 3). As evidenced by 1 H and 31 P NMR, oxazolines 7 were obtained as a mixture of two regioisomers 7 and 7 , in a 66:34 ratio for 7a, while a 65:35 ratio was observed for oxazoline 7c. Oxazolines 7 were purified by flash-column chromatography, allowing the isolation of a single isomer, corresponding to the minor one in the case of 7a+7 a. However, in the case of regioisomeric oxazolines 7c+7 c, the separation of both regioisomers was not possible, and the same 65:35 ratio was obtained after purification by flash-column chromatography. We also tested the ring expansion of N-arylcarbamoyl cyanoaziridines derived from phosphonate 5b and 5d under the optimal conditions. For instance, phosphonatederived oxazolines 7b (R = OEt, R 2 = Ph) and 7d (R = OEt, R 2 = p-MeC 6 H 4 ) were obtained as regioisomeric mixtures after treatment with 5b and 5d, respectively, with 0.2 equivalents of NaI at 60 • C in THF (Table 3, entries 2 and 4). Conversely, the NaI catalyzed ring expansion of N-alkylcarbamoyl cyanoaziridines 5g (R = Ph, R 1 = Me, R 2 = Et) and 5k (R = Ph, R 1 = Et, R 2 = t Bu) to the corresponding oxazolines was not observed, and N-functionalized cyanoaziridines 5g and 5k were recovered instead. A rational mechanism for the formation of oxazoline derivatives 7 can be explained via the initial aziridine ring opening in 5 by an indiscriminate iodide attack to either aziridine carbons C2 or C3. Subsequent ring closure by iodide displacement would afford a mixture of regioisomeric oxazolines 7 and 7 . It seems reasonable to assume that the role of the stereoelectronic nature of N-substituents on the aziridine ring may affect the selectivity of these cyanooxazoline derivatives. Only one regioisomer was formed in the reaction of N-acylcyanoaziridine 2a in the presence of NaI, suggesting the possibility that the N-acyl substituent could exert a neighboring group participation effect, although this does not take place in the case of the N-arylcarbamoyl group.
Several attempts have been carried out in the synthesis of thiazoline derivatives starting from aziridines. It is known that 2-substituted oxazolines or imidazolines can be prepared by the ring expansion of aziridines or benzoylated imidoyl aziridines, respectively [41,45,46], through the Heine reaction. For instance, aziridines undergo ring expansion reactions into oxazolines with Lewis acids [47] and, recently, based on these results, Tepe et al. [48] have described the isomerization of aziridines to oxazolines using BF 3 ·OEt 2 . For this reason, we explored the ring expansion of functionalized N-thiocarbamoyl cyanoaziridine 6a. Initially, we studied the aziridine ring opening under thermal conditions. Thus, N-thiocarbamoyl cyanoaziridine derived from phosphine oxide 6a was heated in refluxing CHCl 3 . Under these conditions, no reaction was observed, and the unreacted starting substrate was recovered. Next, the Heine-type reaction was also studied under nucleophilic conditions by using NaI at 60 • C in THF, and as in the previous case, no satisfactory results were attained.
Likewise, the conversion of aziridine to thiazoline under mild acidic conditions was examined. N-Functionalized cyanoaziridine 6a was treated with both Brønsted acids, such as p-toluenesulfonic acid (PTSA), and Lewis acids, such as ZnCl 2 or BF 3 ·OEt 2 . Only the use of BF 3 ·OEt 2 gave satisfactory results. Hence, when N-thiocarbamoyl cyanoaziridine 6a reacted in the presence of 5 equivalents of BF 3 ·OEt 2 at −70 • C in THF, the formation of 2-aminothiazoline phosphine oxide 8a was detected (Scheme 3). Spectroscopic data confirmed the isomerization of aziridine 6a into 2-aminothiazoline 8a. While the 1 H NMR spectrum of 6a showed a signal for the methyl group at δ H = 2.0 ppm and the methine hydrogen resonated at δ H = 3.8 ppm as a well-resolved doublet ( 2 J PH = 20 Hz), in 2aminothiazolidine 8a these signals appeared at lower fields: δ H = 2.11 and 4.58 ppm as a singlet and a well-resolved doublet ( 2 J PH = 12.8 Hz), respectively. Since it was not conclusively irrefutable that 1 H and 13 C NMR were assigned to the regio-and stereochemistry of compound 8a, the X-ray diffraction analysis not only established the regiochemistry of compound 8a, but also the syn-relationship between the cyano group at the C3 position and the phosphorus moiety at the C2 position of 8a ( Figure 2).
A reasonable mechanism that would explain the formation of 8a is exemplified in Scheme 3. First, BF 3 ·OEt 2 would coordinate with the sulfur atom of cyanoaziridine 6a, thus assisting the ring opening reaction through the N-C3 bond, with the concomitant generation of the most stable carbocation. The cationic intermediate coming from the aziridine with an E-stereochemistry would isomerize, and the ring closure would lead to 2-aminothiazoline 8a as the only regio-and stereoisomer. Continuing with our interest in the synthesis of new nitrogen-containing heterocyclic compounds, finally we examined the one pot reaction of cyanoaziridines 1 with isocyanates in order to obtain new oxazoline derivatives.
For this purpose, phosphorus subtituted cyanoaziridine 1b (R = Ph, R 1 = Et) reacted with phenyl isocyanate in acetonitrile at 60 • C, leading to the formation of iminooxazolidine 9a in low yields (Scheme 4). The addition of 2 equivalents of isocyanate led to 9a in moderate yields (45%), whereas, when the reaction was examined in the presence of KI (30% mol) using 2 equivalents of phenyl isocyanate in acetonitrile at 60 • C, the corresponding iminooxazolidine 9a was obtained in a 62% yield (Scheme 4, Table 4, entry 1). Similarly, cyanoaziridine 1a (R = Ph, R 1 = Me) reacted with p-tolenesulfonyl isocyanate using the same reaction conditions, providing a 55% yield of iminooxazolidine 9b (Scheme 4, Table 4, entry 2). This synthetic methodology was extended to the use of cyanoaziridines derived from phosphonate. Thus, 1c (R = OEt, R 1 = Me) reacted with phenyl isocyanate in the presence of KI in acetonitrile at 60 • C to give iminooxazolidine 9c (Scheme 4, Table 4, entry 3).  A reliable mechanism for the formation of 9 would indicate the addition of an equivalent of isocyanate to cyanoaziridine 1 to obtain the corresponding N-carbamoyl cyanoaziridines 5 (Scheme 4). Then, the regiospecific attack of the iodide ion at the less substitute carbon atom (C2) in aziridines 5 would lead to the ring opening, affording intermediates 10. The former intermediates would attack the carbon center of a second isocyanate equivalent followed by the ring closure to yield iminooxazolidines 9.

Biological Results
The in vitro cytotoxicity of our novel N-functionalized cyanoaziridines 2, 5 and 6 derived from phosphine oxide (R = Ph) and phosphonate (R = OEt), as well as the fivemembered nitrogen-containing heterocycles 3a, 7, 8a and 9 was evaluated by testing their antiproliferative activities against the human cancer cell line A549 (carcinomic human alveolar basal epithelial cells). In order to evaluate the growth inhibition, a cell counting kit (CCK-8) assay was applied. Cell proliferation inhibitory activities as IC 50 values for all the synthesized compounds and chemotherapeutic doxorubicin (DOX) are displayed in Table 5. Likewise, healthy lung cells, such as MRC-5 non-malignant lung fibroblasts were tested to study the selective cytotoxicity [49]. We first examined the nitrogen-substitution effect of the corresponding cyanoaziridines into their cytotoxicity against A549 cell lines. The best result was observed for N-acylated cyanoaziridine 2a derived from phosphine oxide with an IC 50 value of 22.9 ± 1.9 µM (Table 5, entry 2). However, N-acylated cyanoaziridines derived from phosphine oxide 2b and phosphonate 2c (Table 5, entries 3 and 4), as well as N-carbamoyl cyanoaziridines 5a-k ( Table 5, entries [6][7][8][9][10][11][12][13][14][15][16] and N-thiocarbamoyl cyanoaziridines 6a-c ( Table 5, entries 17-19) did not exhibit any toxicity toward the A549 cell line.   Concerning the new 5-membered nitrogen-containing heterocycles derived from the ring expansion of N-functionalized cyanoaziridines against the A549 cell line in vitro, oxazoline derivative 3a showed a IC 50 value of 19.7 ± 2.8 µM (Table 5, entry 5). Conversely, neither the regioisomeric oxazolines 7+7 (Table 5, entries 20-23) nor the 2-aminothiazoline derivative 8a (Table 5, entry 24) displayed any cytotoxicity against the same cell line. Finally, we studied the cytotoxicity effect of iminooxazolidines 9a-c against A549 cell lines. For instance, IC 50 values between 6.2 ± 0.7 and 16.4 ± 1.5 µM were observed, with iminooxazolidine 9c (Table 5, entry 27) as the most effective compound with an IC 50 value of 6.2 ± 0.7 µM. It appears rational to presume that the observed cytotoxic activity in imonooxazolidines 9, which was not observed in oxazolines 7, could be due to the presence of an amide group at the N-3 of the oxazoline ring.
Furthermore, MRC-5 non-malignant lung fibroblasts were tested to study the selective toxicity [49], and none of the synthesized phosphorus substituted N-functionalized cyanoaziridines, 5-membered nitrogen-containing heterocycles, or doxorubicin exhibited any toxicity toward the MRC-5 cell line (see Table 5).

General Experimental Information
Solvents for extraction and chromatography were of a technical grade. All solvents used in reactions were freshly distilled and dried over molecular sieves 4 Å before use. All other solvents and reagents were obtained from commercial sources (Sigma-Aldrich, Spain) and recrystallized or distilled as necessary or were used without further purification. All reactions were performed under an atmosphere of dry nitrogen. Melting points were determined using the Büchi Melting Point B-540 apparatus and were uncorrected. IR spectra were measured on a Nicolet iS10 Thermo Fisher Scientific spectrometer (Thermo Scientific Inc., Waltham, MA, USA) as neat solids. Absorbance frequencies are given at maximum of intensity in cm −1 . High-resolution mass spectra (HRMS) were measured on an Agilent 6530 Accurate-Mass QTOF LC/MS (Santa Clara, CA, USA) by a positive-ion electrospray ionization (ESI) method with a time-of-flight Q-TOF system. Data are reported in the form m/z (intensity relative to base = 100). 1 H (300, 400 MHz), 13 C (75, 100 MHz), and 31 P NMR (120, 160 MHz) spectra were recorded on Varian Unity Plus (Varian Inc., NMR Systems, Palo Alto, CA, USA) or on Bruker Avance 400 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometers, respectively, in CDCl 3 at 25 • C. Chemical shifts (δ H ) are reported in parts per million (ppm) with the internal chloroform signal at 7.24 ppm as the standard for 1 H NMR. Chemical shifts (δ C and δ P ) are reported in parts per million (ppm) with the internal chloroform signal at 77.0 ppm as the standard for 13 C NMR, or the external H 3 PO 4 (50%) signal at 0.0 ppm as the standard for 31 P NMR. All coupling constants (J) values are given in Hz. 13 C NMR spectra were recorded in a broadband decoupled mode from hydrogen nuclei. Distortionless Enhanced Polarization Transfer (DEPT) supported peak assignments for 13 C NMR. The data are reported as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, bs = broad singlet. Chromatographic purification was performed as flash chromatography using commercial grades of silica gel finer than 230 mesh with pressure. Analytical thin layer chromatography (TLC) was performed on precoated Merck silica gel 60 F 254 TLC aluminium plates, and spot visualized with UV light or permanganate stain. Cyanoaziridines 1 were prepared according to procedures in the literature [16].

Experimental Procedure and Characterization Data for Compounds 2-9
General Procedure and Spectral Data for The Addition of 3,5-Dinitrobenzoyl Chloride to Functionalized Cyanoaziridines 3,5-Dinitrobenzoyl chloride (1.4 g, 6 mmol, 1.2 eq) and Et 3 N (2.8 mL, 20 mmol, 4 eq) were added to a 0 • C solution of cyanoaziridine (5 mmol, 1 eq) in CH 2 Cl 2 (25 mL). The reaction mixture was stirred at 0 • C until TLC showed the disappearance of the starting cyanoaziridine. The crude product was washed three times with a saturated NaCl solution (15 mL) and water (15 mL) and extracted with CH 2 Cl 2 (15 mL). The organic layers were dried over anhydrous MgSO 4 , filtered and concentrated to dryness in vacuum conditions, and the resulting residue was purified by crystallization from Et 2 O/pentane or washed with pentane.

General Procedure and Spectral Data for Compound 3a
To a stirred solution of N-functionalized cyanoaziridine 2a (5 mmol, 1 eq) in THF (15 mL), NaI (0.02 g, 1 mmol, 0.2 eq) was added dropwise. The mixture was heated at 60 • C for 24 h until TLC showed the disappearance of the starting cyanoaziridine. The reaction mixture was concentrated to dryness in vacuum conditions to remove THF. The crude product was washed three times with water (15 mL) and extracted with CH 2 Cl 2 (15 mL). The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated to dryness in vacuum conditions. The crude product was purified by flash-column chromatography. Method A. To a 0 • C solution of cyanoaziridine (5 mmol, 1 eq) in CH 2 Cl 2 (25 mL) the corresponding isocyanate (6 mmol, 1.2 eq) was added dropwise. The reaction mixture was allowed to reach room temperature and stirred for 6-24 h. The crude products were concentrated to dryness in vacuum conditions and were purified by crystallization.
for 24 h until TLC showed the disappearance of the starting N-functionalized cyanoaziridine. The crude product was washed three times with water (15 mL) and extracted with CH 2 Cl 2 (15 mL). The organic layers were dried over anhydrous MgSO 4 , filtered, and concentrated to dryness in vacuum conditions, and the resulting residue was purified by flash-column chromatography.