Synthesis and Anticancer Evaluation of Novel Derivatives of Isoxazolo[4,5-e][1,2,4]triazepine Derivatives and Potential Inhibitors of Protein Kinase C

Abstract

In the present study, using Thorpe’s reaction with Gewald’s modification, 4-acetylamino-5-acetyl or 5-benzoyl 3-carboxamide compounds 3 or 4 were obtained. From these compounds, two series of compounds (5, 7, and 9 and 6, 8, and 10) were obtained with 98% hydrazine. Compounds 6, 7, 8, and 9 were then reacted with the appropriate aldehydes to afford a series of new isoxazole derivatives (11–18, 27–36, and 37–41) and the main compounds, 19–26 and 42–45, were isoxazolo[4,5-e][1,2,4]triazepine derivatives. The anticarcinogenic activities of selected compounds were tested on six lines of cancer cells, and their activities were compared with the relevant concentrations of the anticarcinogenic drugs cisplatin and doxorubicin in IITD PAN. Several compounds were tested on 60 lines of cancer cells by the NCI (Bethesda, MD, USA). The cyclization of compound 12 into derivative 46 was also carried out. Compound 21 showed extremely high antitumor activity.

Introduction

Continuing the synthesis of heterocyclic compounds in the derivative group isoxazole and hydrazine, we obtained derivatives isoxazolo[4,5-e][1,2,4]triazepine of a new heterocyclic system on two synthetic routes, namely, by reaction rearrangement of O: Dimroth and by cyclization of relevant derivatives isoxazole. The isoxazolo triazepine heterocyclic system, which possesses five heteroatoms (one oxygen atom and four nitrogen atoms) has 12 isomers. The isomer of interest in this study, [4,5-e][1,2,4], and its derivatives have not yet been described in the chemical literature. The distribution of three heteroatoms in this isomer is similar to that in 9H-purines or pentostatin. Therefore, we expect that the anticarcinogenic activity of these compounds, as shown in Tables 1 and 2, may be a very interesting biological mechanism of action. We based our tests on a comparison of the chemical structure of our compounds with the structure of naturally occurring phorbol diesters and mezerein, Figure 1. They are strong PKC activators.¹⁻⁴ The PKC family consists of 13 isoenzymes, and the distribution of some of the isoenzymes is closely related to the type of tissue. All PKC have a single polypeptide structure that contains the catalytic domain region and the regulatory domain region. The domains have binding sites for activators and inhibitors. In the catalytic domain, adenosine 5′-triphosphate is the endogenous activator. The regulatory domain has diacylglycerol (DAG) as the endogenous domain. The pathological role of individual PKC isoenzymes is associated with their long hyperactivity which leads to cancer or other disorders. There are certain naturally occurring toxic compounds, such as the rottlerin (mallotoxin) from the plant Mallotus philippinensis and the staurosporine—an alkaloid of bacterial origin. The chemical structures of these compounds were the basis for the design of numerous inhibitors through various modifications. There are rather few studies describing the syntheses of these compounds which were proposed as inhibitors of catalytic domain,^{6,17,18,20−23}Figure 2. There have also been attempts to obtain PKC, inhibitors using phorbol diesters.²⁰⁻²³ The naturally occurring phorbol diesters in the Croton tiglium (Euphorbiaceae) are strong and have long-lasting binding to the regulatory domain of protein kinases C.^{1−7,16,32−34}

Table 1.

^a,c

	mean IC50 ± SD [μg/mL]
compound	LoVo	LoVoDx	MV-4-11	A549	MCF-7	Balb/3T3
21	0.16 ± 0.04	0.24 ± 0.04	0.15 ± 0.01	NT	NT	NT
22	0.51 ± 0.12	0.45 ± 0.11	0.21 ± 0.01	2.70 ± 1.23	NT	NT
24	3.61 ± 0.32	3.59 ± 0.32	2.72 ± 0.28	0.82 ± 0.41	NT	NT
37	[40.04]b± [2.82]	[29.45]b± [14.98]	98.45 ± 1.25	[25.44]b± [10.11]	NT	NT
38	23.55 ± 6.55	27.22 ± 6	28.62 ± 6.11	38.22 ± 11.4	NT	NT
39	35.11 ± 2.11	42.98 ± 6.41	40.25 ± 16.33	61.58 ± 2.11	NT	NT
40	24.00 ± 0.54	55.70 ± 11.21	49.22 ± 13.7	35.66 ± 2.65	NT	NT
41	22.14 ± 2.98	35.54 ± 2.62	32.93 ± 0.11	39.73 ± 7.8	NT	NT
46	NT	NT	3.29 ± 0.29	5.15 ± 0.34	3.29 ± 0.29	5.36 ± 0.43
cisplatin	1.18 ± 0.09	0.76 ± 0.1	0.49 ± 0.01	0.90 ± 0.22	1.57 ± 0.46	1.83 ± 0.22
doxorubicin	0.10 ± 0.03	3.23 ± 0.77	0.03 ± 0.01	0.05 ± 0.01	NT	NT

^aNT—not tested.

^bMean proliferation inhibition ± SD measured for highest concentration used (100 μg/mL).

^cData from HIIET. The values below 10 μM indicates considerable antiproliferative activity.

Table 2. Results of the in Vitro Screening of 60 Human Tumor Cell Lines^a.

	panel/cell line	–5.0	–5.0	–6.0	–5.0	–5.0	–5.0	–5.0	–5.0
		log10 concentration units: molar
		percent growth
	compound	11	17	21		23	27	34	40
leukemia	CCRF-CEM	122	86	14	13	53	109	51	133
	K 562	84	126	18	14	21	98	117	81
	MOLT-4	113	68	33	28	54	137	122	145
	RPMI-8226	130	103	50	10	74	94	114	113
	SR	94	119	11	7	34	50	102	127
nonsmall-cell lung cancer	A549/ATCC	123	95	57	39	75	133	103	114
	EKVX	102	111	29	3	57	91	99	116
	HOR-92	95	91	66	6	56	88	56	97
	NCI-M-226	103	99	42	39	91	112	103	113
	NCI-H322 M	119	120	38	25	80	111	102	104
	NCI-H 460	111	101	12	2	88	106	108	106
	NCI-H 522	91	88	8	7	63	117	67	107
colon cancer	COLO-205	110	113	2	–30	86	112	85	110
	HCC-2988	115	117	48	–23	–28	107	90	119
	HCT-116	102	99	57	36	65	120	94	115
	HCT-15	105	103	25	14	90	98	124	102
	HT-29	120	109	–37	–37	76	107	103	113
	KM-12	98	122	39	22	57	181	103	99
	SW-620	108	106	30	28	44	115	104	104
CNS cancer	SF-268	100	114	10	–2	84	106	96	105
	SF-295	108	80	–18	–34	76	123	110	121
	SF-539	95	99	–20	–49	29	102	90	99
	SNB-19	96	74	16	15	77	104	79	109
	SNB-75	122	108	83	53	53	106	93	104
	U-251	106	105	36	30	86	99	114	95
melanoma	LOX IMVI	91	101	41	28	80	104	112	99
	MALME-3M	108	93	55	37	70	122	92	109
	M-14	109	112	–9	–12	73	111	103	116
	MDA-MB-435	110	108	–51	–54	72	106	101	112
	SK-MEL-28	42	118	83	75	62	111	115	103
	SK-ME-5	96	97	4	–7	48	102	90	100
	UACC-257	76	83	71	72	66	31		32
	UACC-62	102	98	27	21	3	114	105	111
ovarian cancer	OVCAR-3	104	122	51	47	83	118	109	146
	OVCAR-4	123	109	68	53	75	111	109	109
	OVCAR-5	113	104	38	10	77	119	100	123
	OVCAR-8	41	45	42	25	73	49	28	47
	NCI/ADR-RES	117	105	–21	–47		115	108	112
	SK-OV-3	106	75	23	18		108	87	109
renal cancer	786-O	196	96	27	26	81	97	88	97
	A-498	115	109	22	12	57	126	128	116
	ACHN	110	110	37	31	88	99	148	109
	RXF-393	112	92	–12	–15	94	124	72	123
	SN 12 C	114	108	42	37	85	121	135	101
	TK-10	119	119	31	16	87	114	113	135
	UO-31	62	–10	58	46	84	93	–29	83
prostate cancer	PC-3	106	84	38	33	80	103		110
	DU-145	110	130	–12	–25	60	180	101	114
breast cancer	MCF-7	98	107	9	0.0	89	84	84	88
	MDA-MB-231/ATCC		113	51	29	60	134	105	110
	HS 578T	117	112	34	33	46	113	134	104
	BT-549	102	106	24	–11	55	105	100	97
	MDA-MB-435					6

^aData of the tested compounds were obtained from the National Cancer Institutes.¹⁸ The values higher than 40% are not considered significant inhibition of the growth of tumor cells. The values below 40% indicate considerable antitumor activity. The values minus the mean total inhibition growth of the tumor cells and the observed % mortality of the tumor cells are given for each compound. Therefore, we explored the binding of active compounds 21 and 23 to adenosine receptor A3, but the results were negative.

Figure 1.

Naturally occurring TPA (phorbol diester) and mezerein in laboratory tests have shown carcinogenic activity.¹⁻³ However, our compound 24 with a similar structure but devoid of the ester group showed anticancer activity (Table 1).

Figure 2.

Compounds inhibit enzymatic activity by binding to the catalytic domain of kinase PKC.^{6,17,18,20−23}

Chemistry

Results and Discussion

These naturally occurring compounds also have a five-membered ring condensed with a seven-membered ring in the “southern” part of their structure. The core structure of our compounds [4,5-e][1,2,4] resembles those of phorbol esters in the “southern” part of their structure,¹⁻⁴Figure 1. The heteroatoms present in this part, namely, the C=O group in position 3 and the OH group in position 9 are responsible for the long-lasting binding of phorbol diesters to the C1 and C2 regions of the regulatory domain of PKC.^22,23,35 However, the part of the adjacent six-membered ring the (northern) group has two substituents—acyl groups in positions 12 and 13. Those two acyl groups make up the northern part of phorbol esters and mimic DAG structure and bind with the part of protein kinase C regulatory domain to which endogenous ligand DAG is physiologically binding, but only very briefly. In contrast, phorbol esters by long-term binding to regulatory domain are responsible for carcinogenesis.

Under physiological conditions, those two acyl group-binding domain is transiently occupied by endogenous ligands of the regulatory domain of DAG; thus, extended exposure to phorbol esters, which mimic DAG, can cause cancer.^8−14,36,37

Our compounds imitate only structure of “southern” phorbol, because it has an oxygen atom in position 1 that can form hydrogen bonds in position similar to the C=O group in position 3 of phorbol ester, and in our compounds, there is an NH group in position 4 similar to an OH group in position 9 which is a key feature in the binding of phorbol to the kinase regulatory domain, Figure 3. Our compounds do not have an acyl group at position 5 which means that they cannot imitate DAG; however, they can tightly bind to the PKC regulatory domain, and because of their alkyl-, not acyl-derived substituent at position 5, they can block DAG from accessing the regulatory domain for long enough. Of course, in tumor cells, these compounds can be classified as inhibitors of the PKC regulatory domain. We have verified our idea not by using isolated protein kinase C but on the live tumor cells (60 cell lines) from different tissues.¹⁶ This method gives concrete and more useful results for extended research.

Figure 3.

Superposition of compound 24 (red) and phorbol diester. X shows ideally covering atoms of two compounds.

The protein kinase C inhibitors are used where excessive nonphysiological stimulation has caused lesions, for example, in diabetes, heart diseases, and cancer.³⁸⁻⁴⁰

The synthetic route to access 6-acetyl-8-methyl-5-alkyl or aryl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepines and 6-acetyl-8-phenyl-5-alkyl or aryl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2.4]triazepines 19–26 and 1,2-oxazole (=isoxazol) derivatives 11–18, 27–36, and 37–41 as well 43–45 is outlined in Scheme 1.

Scheme 1. Synthesis and Structures of Intermediates Compounds 1–4 Are Described in Refs^24,28.

(a) Comp. 3 or 4, absolute ethanol, hydrazine (98%), under N₂, 60 °C, TLC analysis, CHCl₃, isopropyl alcohol, 12 h rt solid filtration yield crude compound of 5 (dry-clean), from the filtrate by column chromatography to obtain compounds 7 and 9. (b) Comp. 6 (10 mmol, appropriate aldehyde (11 mmol), absolute ethanol, rt 8 h, column chromatography, to lead to compounds 11–18. (c) Comp. 7 or 8 (0.1 mmol), anhydrous, one drop 20% HCl, appropriate aldehydes (0.1 mmol), rt NaHCO₃, column chromatography. (d) Comp. 9 (0.1 mmol), absolute ethanol, appropriate aldehydes, (0.11 mmol), 60 °C, 8 h. (e) Comp. 12 or 13 or 16–18 (0.03 mmol), mixture of Ac₂O 1:1 anhydrous pyridine, rt 15 h, ice water. (f) Comp. 31 or 33–34 or 21 (0.01 mmol), mixture Ac₂O 1:1 anhydrous pyridine, ethyl ether, anhydrous methanol.

5-Acetyl-4-amino-1,2-oxazole-3-carboxamide (1) and 4-amino-benzoyl-1,2-oxazole-3-carboxamide (2) were prepared using a Thorpe reaction according to Gewald et al.,²⁴ and these compounds were converted to 4-acetyl derivatives 3 and 4. Compound 3 was heated with excess 98% hydrazine to give a mixture of three compounds, 5, 7, and 9. Compound 4 was similarly heated with excess 98% hydrazine, which also led to a mixture of three compounds 6, 8, and 10. After separation of the mixtures into pure compounds by column chromatography and multiple crystallizations, three pairs of structurally similar compounds, 5/6, 7/8 and 9/10 were obtained. Compounds 3 and 4 despite having acetyl and benzoyl groups at position 5 were reacted with a hydrazine exchange with the amide groups in positions 3 to afford −NH–NH₂ moieties; thus, hydrazides 5 and 6 were formed. The ketone moieties of 7 and 8 were reacted with hydrazine to form the hydrazones (compounds 3 and 4), and then, in the same reaction vessel, the transacetylation between the acetyl amine at position 4 of compounds 3 and 4 and the newly formed NH₂ group of the hydrazone was achieved.

Compounds 9 and 10 were formed as the hydrazones of compounds 3 and 4. Compounds 6, 7, 8, and 9, which were formed in good yields, were condensed with the appropriate aliphatic or aromatic aldehyde in the presence of a catalytic amount of p-toluene sulfonic acid. The condensation of 6 with the appropriate aldehydes gave hydrazine–hydrazones 11–18, and compound 9 gave hydrazone–hydrazone derivatives, compounds 27–36Scheme 1.

Compounds 7 and 8 (acetylhydrazones) may exist as geometrical isomers (E/Z) with respect to the C=N double bonds and as rotamers (cis/trans) with respect to the amide (N–C=O) bonds. As described by Palla et al.,²⁵ and Syakaev²⁶et al., only the E form occurs because of the hindered rotation around the imine bond. The cyclization of 7 and 8 with aliphatic or aromatic aldehydes formed target compounds 19–26 which are isoxazolo[4,5-e][1,2,4]triazepine derivatives. The chemical structures of these compounds were confirmed by X-ray crystallography, as shown in Figure 4. Several compounds 12, 13, and 16–18 among methylidene carbohydrazide were cyclized with a 1:1 mixture of pyridine and acetic anhydride,²⁷ directly via O. Dimroth-type rearrangement of isoxazolo[1,3,4] oxadiazole derivatives 37–41 were obtained, and they showed the expected anticancer effects. Compounds 31, 33, and 34, from methylidene hydrazinylidene (27–36) were cyclized under the same conditions to afford via Dimroth-type rearrangement of compounds 42–44. Compound 21 under the same condition gave compound 45. The cyclization of compound 12 led to compound 46 (Scheme 2).

Figure 4.

X-ray molecular structure of compound 21 along with the numbering scheme. Displacement ellipsoids are shown at the 30% probability level.

Scheme 2. Compound 46: Compound 12 Absolute Ethanol, Acetylhydrazine, 60 °C, 8 h, Fresh Distilled Isobutyl Aldehyde 60 °C, 5 h, 1 mg of p-Toluene Sulfonic Acid, Column Chromatography, Crystallizations, Isopropyl Alcohol.

Biological Activity

Antitumor Activity (Table1)

The antiproliferative activities of nine compounds (21, 22, 24, 37, 38, 39, 40, 41, and 46) were assessed in vitro on five diverse human tumor cell lines representing colon cancer (LoVo and LoVoDx), leukemia (MV-4-11), nonsmall-cell lung cancer (A549), breast cancer (MCF-7), and murine normal fibroblasts Balb/3T3. The widely used cytostatics cisplatin and doxorubicin were used as reference agents. We observed significant structure–activity relationships with IC₅₀ differences exceeding 100-fold in some cell lines. Compound 37 was the least active against all the tested cell lines. Isoxazolotriazepine derivatives 21, 22, and 24 proved to be the most active. The two most active compounds, 21 and 22, were more than twice as active as cisplatin on LoVo, LoVoDx, and MV-4-11 cell lines. Compound 24 showed the same activity as cisplatin on A549 cells and as doxorubicin on LoVoDx. Isoxazole derivatives 37, 38, 39, and 40 showed lower activities on all cell lines. The activity of compound 46 was twofold lower than that of cisplatin on MCF-7.

As shown in Table 2, only seven of the synthesized compounds (11, 17, 21, 23, 27, 34, and 40) were analyzed by the National Cancer Institute (Bethesda, MD, USA). The in vitro antitumor activities of these compounds were screened against 60 different human tumor cell lines. Their antitumor activities are reported as the percentage of growth of the treated cells, which represented leukemia, nonsmall-cell lung cancer, colon cancer, central nervous system (CNS) caner, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer, following a single dose of 10 mol, and compound 21 was tested at a dose of 1 μmol. Values in Table 2 higher than 40% are not indicative of significant cancer cell growth inhibitors. Values lower than 40% indicate significant anticancer activity. Only negative percentages indicate the full inhibition of tumor cell growth by the compound, and these values indicate the percent of dead tumor cells. The outstanding activities of our compounds were demonstrated by compound 21, that is, 6-acetyl-8-phenyl-5-(propan-2-yl)-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide. At a dose of 10^–5 mol (10 μmol), growth inhibition was observed against colon cancer cells (Colo-205, HCC-2988, and HT-29), neoplastic cells of the CNS (9F-296 and SF-539), melanoma cells (M-14, MDA-MB-435, and SK-ME), ovarian cancer cells (NCJ/ADR-RES), renal cancer cells (RXF-393), prostate cancer cells (DV), and breast cancer cells (BT-549). The results observed at a dose 10 times lower (at 10^–6 mol = 1 μmol) are particularly interesting because the percentages of inhibition are slightly lower but still noticeable in the abovementioned cancer cell lines. Compound 28, another compound from the same group as that of isoxazolo triazepine 23, only significantly inhibited the colon cancer cell line HCC-2988, and it showed weak inhibition (below 40%) against leukemia cells (K-562 and SR), CNS cancer cells (SF-539), melanoma (UACC-62), and breast cancer cells (MDA-MB-435). Isoxazole derivatives 17, 27, 34, and 40 showed moderate activities. 17 and 34 both only significantly inhibited UO-31 cells. The tested compounds did not show cytotoxic effects against the remaining 59 cancer cell lines, and in some lines, they even stimulated cancer cell growth. This selective action on one or several types of cancer as well as the growth-enhancing effects may indicate the mechanism of action of these compounds against cancer. The inhibition of one or more of the 13 different PKC isoesters, which selectively occur^15,38,40 in the relevant tissues, may be involved.

X-ray Crystallographic Analysis of 21

Compound 21 crystallizes in the centrosymmetric, monoclinic I2/a space group with one molecule in the asymmetric unit (Figure 4). The 5,6-dihydrotriazepine ring is not planar, and the N6 and N7 atoms deviate from the plane of the remaining atoms (N4, C5, C8, C9, and C10) by 0.90(1) and 0.48(1) Å, respectively. The isoxazole ring is almost coplanar with the plane formed by N4, C5, C8, C9, and C10, and the dihedral angle is equal to 2.6(1)°. In turn, the amide bound to C3 is coplanar with the isoxazole ring, and their dihedral angle is 1.9(1)°. The intramolecular hydrogen bond between N4–H4···O12 [H···A 2.29 Å, D···A 2.8615(17) Å, D–H···A 122°] supports the coplanarity of these rings. In contrast, the phenyl ring bound to C8 is rotated with respect to the mean plane of N4, C5, C8, C9, and C10 by 61.3(1)°. The molecule possesses a chiral center at C5, but because it crystallizes in the centrosymmetric group, the mixture is racemic.

The crystal structure is stabilized by intermolecular hydrogen bonds. In addition to intramolecular H-bonds, the H4 atom is involved in an N4–H4···O12ⁱ interaction [H···A 2.14 Å, D···A 2.9815(17) Å, D–H···A 159°, (i) −x + 3/2, y, −z + 1] to produce a dimer. These dimers are connected into chains propagating along the a axis (Figure 5) through H bonds formed by the NH₂ group of the amide [N13–H13A···O12ⁱⁱ H···A 2.13 Å, D···A 2.9573(16) Å, D–H···A 157°, (ii) −x + 2, −y, −z + 1; N13–H13B···O12ⁱⁱⁱ H···A 2.06 Å, D···A 2.8912(18) Å, D–H···A 158°, (iii) x + 1, y, z].

Figure 5.

Hydrogen-bonded chain connecting molecules of 21. Hydrogen atoms not involved in the H-bonds are omitted for clarity. Symmetry codes and geometric parameters are presented in the text in the Methods Section at X-ray Diffraction Studies.

Conclusions

Many authors³⁻⁷ have shown that endogenous DAG binds very tightly to the PKC kinase regulatory domain, causing cancer. The mechanism of the effect of phorbol esters involves the irreversible binding of the “southern” part of their chemical structure with the PKC kinase regulation domain and its “northern” diester part imitating the double ester in DAG, and these interactions sustain the physiological activity of cells for a long time, leading to cancers.^8−14,41,42 Compound 21 is structurally similar to the “southern” part of phorbol esters (the ring system and heteroatoms), allowing it to form hydrogen bonds and irreversibly bind the PKC kinase regulatory domain, but it lacks the diester fragment and has an isopropyl group that can effectively block the access of DAG to the regulatory domain in tumor cells. Blocking the physiological function of tumor cells can inhibit cell growth and cause death (Table 2).

Compound 21 has been brought to phase II trials by the NCI Biological Commission (as 1 out of 10,000) to the next institute for further research, which confirmed the first NCI results. At that time, the mechanism of action of compound 21 was not known. Compound 21 has been patented.²⁸

Experimental Section

Chemistry

Melting points were determined on a Boethius apparatus and are uncorrected. The IR spectra (KBr) were recorded on a Jasco FT-IR 420 Fourier spectrometer and are reported in cm^–1. The ¹H NMR and ¹³C NMR spectra were recorded on a Bruker ARX 300 MHz (Bruker Analytical, Karlsruhe, Germany Bruker AG, Fallanden, Switzerland) instrument using dimethyl sulfoxide-d₆ (DMSO-d₆) as the solvent. All chemical shifts (δ) are reported in parts per million down field relative to the chemical shift of tetramethylsilane. Reaction progress and the purity of the obtained compounds were monitored by thin-layer chromatography (TLC) on Merck silica gel plates (Merck a F₂₅₄, Darmstadt). Column chromatography separations were performed using silica gel columns 60 (0.063–0.200 or 0.040–0.063 mm, Merck). Elemental analyses were performed on a PerkinElmer 2400 analyzer (Waltham, MA, USA), and for the new compounds, the results were within ±0.1% of the theoretical values. Mass spectra were determined on a compact mass spectrometer coupled with a liquid chromatograph from Bruker Daltonics. The structure of 21 was determined by X-ray crystallography. The chemicals and reagents for syntheses were obtained from Alfa Aesar (Karlsruhe, Germany) and Chempur (Piekary Sl. Poland).

The syntheses of starting compounds 1–4 are described in ref (24). The general procedures for the syntheses of compounds 5, 7, and 9 from 4-acetamido-5-acetyl(1,2)oxazole-3-carboxamide (3) are described below.

To a solution of compound 3 (0.03 mol, 6.33 g) in absolute ethanol (100 mL) was added hydrazine (98%, 1.9 g), and the reaction mixture was heated at reflux (or air nitrogen) until the starting material was consumed (TLC monitoring). The volume was reduced to half by evaporation, and the mixture was kept at room temperature overnight. The precipitate was separated by filtration and washed thrice with 30 mL of methanol to yield crude compound 5 (1.2 g). The filtrate contained two other compounds and was concentrated to dryness. The residual solid was purified by column chromatography (CHCl₃/isopropanol 7:1) to give pure compound 7 and compound 9.

5-Acetyl-4-amino-1,2-oxazole-3-carbohydrazide (5)

The crude compound obtained and described in the procedure from compound 3 in the amount of 1.2 g was recrystallized from ethanol to yield 0.85 g of pure material (15.39%). mp 194 °C; IR (cm^–1): 3440, 3355, 3300, 3200 (NH), 1680, 1643 (C=O), 1610 (C=N); ¹H NMR (DMSO-d₆): δ 2.04 (3H, s, −CH₃), 4.72 (2H, br, −NH₂), 6.35 (2H, br, −NH₂), 10.22 (1H, s, O=C–NH−); ¹³C NMR (300 MHz, DMSO-d₆): δ 10.7, 114.7, 136.3, 147.4, 150.4, 159.2, 166.1; MS (ESI) (m/z): [M + H]⁺ 185.0669. Anal. Calcd for C₆H₈N₄O₃ (184.15): C, 39.13; H, 4.38; N, 30.42. Found: C, 39.20; H, 4.51; N, 30.15.

5-[-1-(2-Acetylhydrazinylidene)ethyl]-4-amino-1,2-oxazole-3-carboxamide (7)

Derivative 7 was obtained as a white solid and was recrystallized from dioxane to yield 1.85 g (27.4%) of material. mp 210 °C; IR (cm^–1): 3495, 3394, 3200 (N–H), 1683, 1672 (C=O), 1632 (C=N); ¹H NMR (DMSO-d₆): δ 2.04 (3H, s, −CH₃), 2.26 (3H, s, −CH₃), 5.56 (2H, br, NH₂), 7.82 (1H, s, O=C–NH), 8.06 (1H, s, O=C–NH), 10.68 (1H, s, O=C–NH–N=); ¹³C NMR (300 MHz, DMSO-d₆): δ 12.8, 23.0, 112.1, 134.4, 156.3, 159.0, 168.8; MS (ESI) (m/z): [M – H]⁺ 224.0778. Anal. Calcd for C₈H₁₁N₅O₃ (225.20): C, 42.67; H, 4.92; N, 31.10. Found: C, 42.29; H, 4.81; N, 31.33.

4-Acetamido-5-(1-hydrazinoethyl)-1,2-oxazole-3-carboxamide (9)

Compound 9 was obtained as a white solid and was recrystallized from ethanol to give 3.17 g (46.55%) of the desired product. mp 218 °C; IR (cm^–1): 3482, 3364, 3212 (N–H), 1683 (C=O), 1594 (C=N), 1300, 1220; ¹H NMR (DMSO-d₆): δ 2.0 (3H, s, CH₃), 2.30 (3H, s, −CH₃), 5.22 (2H, br, N–NH₂), 7.88 (1H, s, HN–C=O), 8.18 (1H, s, HN–C=O), 10.12 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 12.8, 23.0, 111.4, 131.0, 156.0, 161.0, 169.5; MS (ESI) (m/z): [M – H]⁺ 224.0778. Anal. Calcd for C₈H₁₁N₅O₃ (225.20): C, 42.67; H, 4.92; N, 31.10. Found: C, 42.69; H, 4.71; N, 31.12.

General Procedure for the Synthesis of Compounds 6, 8, and 10 from 4-Aacetamido-5-(phenylcarbonyl)-1,2-oxazole-3-carboxamide (4)

To a solution of compound 4 (0.02 mol, 5.46 g) in 100 mL of absolute ethanol was added hydrazine (98%, 1.28 g), and the reaction mixture was heated at reflux (in air or N₂) until TLC analysis showed the absence of the starting material (usually 6 h). The solvent was evaporated to give a final volume of approximately 50 mL, and the residue was kept at room temperature overnight. The resulting precipitate was collected by filtration and washed with methanol to yield 3.02 g of crude 6. The filtrate was concentrated under reduced pressure to dryness, and the residues were purified by column chromatography (CHCl₃/isopropanol, 9:1) to give pure compounds 8 and 10.

4-Amino-5-(phenylcarbonyl)-1,2-oxazole-3-carbohydrazide (6)

Crude compound 6 was recrystallized from ethanol to yield 2.9 g (50.3%) of the desired product. mp 211 °C; IR (cm^–1): 3482, 3421, 3364, 3200 (N–H), 1683, 1642 (C=O), 1610 (C=N), 1550, 1350; ¹H NMR (DMSO-d₆): δ 4.69 (2H, br, NH–NH₂), 6.35 (2H, br, NH₂), 7.56–8.07 (5H, m, phenyl), 10.28 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 117.0, 125.1, 125.6, 127.3, 128.0, 128.3, 129.5, 136.6, 153.6, 155.3, 160.3, 168.3; MS (ESI) (m/z): [M – H]⁺ 245.0669. Anal. Calcd for C₁₁H₁₀N₄O₃ (246.22): C, 53.66; H, 4.09; N, 22.75. Found: C, 53.34; H, 3.92; N, 22.52.

5-[(2-Acetylhydrazino)(phenyl)methyl]-4-amino-1,2-oxazole-3-carboxamide (8)

Compound 8 was obtained as a white solid following recrystallization from dioxane (1.8 g, 31.14%). mp 191 °C; IR (cm^–1): 3485, 3422, 3200, 3193 (N–H), 1685, 1670 (C=O), 1610 (C=N), 1580, 1355; ¹H NMR (DMSO-d₆): δ 1.9 (3H, s, −CH₃), 5.71 (2H, br, −NH₂), 7.40–7.59 (5H, m, Ar), 7.86 (1H, s, HN–C=O), 8.10 (1H, s, HN–C=O), 10.52 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 22.1, 112.2, 127.8, 128.0, 128.3, 128.5, 129.6, 131.5, 155.9, 160.3, 161.4, 168.7; MS (ESI) (m/z): [M + H]⁺ 288.1091. Anal. Calcd for C₁₃H₁₃N₅O₃ (287.27): C, 54.35; H, 4.56; N, 24.38. Found: C, 54.01; H, 4.41; N, 24.61.

4-Acetamido-5-[hydrazinylidene(phenyl)methyl]-1,2-oxazole-3-carboxamide (10)

Compound 10 was obtained as a light yellow solid (0.55 g, 9.5%) via recrystallization from ethanol. mp 214 °C; IR (cm^–1): 3465, 3368, 3245, 3200 (N–H), 1685, 1665 (C=O), 1595 (N–H); ¹H NMR (DMSO-d₆): δ 2.28 (3H, s, −CH₃), 5.1 (2H, br, N–NH₂), 7.44–7.51 (5H, m, Ar), 7.89 (1H, s, HN–C=O), 8.21 (1H, s, HNC=O), 10.09 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 22.2, 112.3, 127.7, 128.1, 128.3, 128.6, 129.5, 132.0, 156.2, 161.0, 162.2, 169.4; MS (ESI) (m/z): [M + H]⁺ 288.1091. Anal. Calcd for C₁₃H₁₃N₅O₃ (287.37): C, 54.35; H, 4.56; N, 24.38. Found: C, 54.70; H, 4.66; N, 24.69.

General Procedure for the Preparation of Compounds 11–17, Derivatives of N′-Hydrazides^25,26

An approximately equimolar amount (1.1 mmol) of the appropriate aliphatic or aromatic aldehyde was added to a solution of hydrazide 6 (1 mmol) in 25 mL of absolute ethanol in the presence of a catalytic amount of p-toluene sulfonic acid. The reaction mixture was stirred for 4–6 h at room temperature and then poured into cold water. The formed precipitate was separated by filtration, washed thrice with 20 mL of water, and purified by recrystallization.

4-Amino-5-benzoyl-N′-[(1E)-ethylidene]-1,2-oxazole-3-carbohydrazide (11)

Derivative 11 was obtained as a white solid by the condensation of 6 with acetaldehyde and recrystallization from ethanol. 81% yield; mp 180 °C; IR (cm^–1): 3495, 3365, 3240 (N–H), 1660, 1645 (C=O), 1600 (C=N), 1280, 710; ¹H NMR (DMSO-d₆): δ 1.2 (3H, t, −CH₃), 2.1 (2H, q, CH₂), 6.38 (2H, br, −NH₂), 7.56–8.10 (6H, m, Ar and =CH), 12.01 (1H, s, O=C–NH–N=); ¹³C NMR (300 MHz, DMSO-d₆): δ 18.9, 112.0, 128.5, 129.2, 129.3, 133.5, 136.5, 138.7, 147.2, 147.7, 151.8, 155.8, 180.7; MS (ESI) (m/z): [M + H]⁺ 273.0982. Anal. Calcd: for C₁₃H₁₂N₄O₃ (272.25): C, 57.35; H, 4.44; N, 20.58. Found: C, 57.05; H, 4.53; N, 20.23.

4-Amino-N′-[(1E)2-methylpropylidene]-1,2-oxazole-3-carbohydrazide (12)

Derivative 12 was obtained as a white solid from the condensation of 6 with isobutyl aldehyde and recrystallization from ethanol. 84% yield; mp 169 °C; IR (cm^–1): 3495, 3360, 3260 (N–H), 1657, 1643 (C=O), 1600, 1550, 1280, 710; ¹H NMR (DMSO-d₆): δ 1.05 (3H, d, CH₃), 1.07 (3H, d, CH₃), 2.47–2.57 (1H, m, CH), 6.00 (2H, br, −NH₂), 7.56–8.07 (6H, m, Ar + =CH), 11.96 (1H, s, O=C–NH–N=); ¹³C NMR (300 MHz, DMSO-d₆): δ 12.6, 12.8, 19.4, 113.3, 127.9, 128.6, 128.9, 129.2, 129.3, 132.9, 136.6, 139.0, 147.8, 148.9, 151.8, 155.9, 180.5; MS (ESI) (m/z): [M + H]⁺ 301.1286. Anal. Calcd: for C₁₅H₁₆N₄O₃ (300.31): C, 59.99; H, 5.37; N, 18.66. Found: C, 59.65; H, 5.51; N, 18.43.

4-Amino-5-benzoyl-N′-[(1E)-ethylbutylidene]-1,2-oxazole-3-carbohydrazide (13)

Derivative 13 with 2-ethylbutyraldehyde was obtained in 80% yield as a white solid following recrystallization from ethanol. mp 179 °C; IR (cm^–1): 3495, 3365, 3230 (N–H), 1665, 1635 (C=O), 1600 (C=N), 1550, 1530, 720; ¹H NMR (DMSO-d₆): δ 0.83 (3H, t, CH₃), 0.88 (3H, t, CH₃), 1.42–1.49 (4H, m, 2 × CH₂), 2.08 (1H, m, CH), 6.39 (2H, br, NH₂), 7.58–8.07 (6H, m, Ar + =CH), 11.99 (1H, s, O=C–NH–N=); ¹³C NMR (300 MHz, DMSO-d₆): δ 13.8, 14.0, 14.2, 21.7, 21.8, 22.5, 25.0, 25.1, 28.5, 30.8, 30.9, 32.4, 116.5, 127.6, 128.6, 128.3, 128.5, 129.3, 132.9, 136.6, 147.9, 148.9, 155.4, 158.3, 180.2; MS (ESI) (m/z): [M + H]⁺ 329.1608. Anal. Calcd: for C₁₇H₂₀N₄O₃ (328.36): C, 62.18; H, 6.14; N, 17.06. Found: C, 62.50; H, 6.38; N, 17.41.

4-Amino-5-benzoyl-N′-[(1E)-phenylmethylidene]-1,2-oxazole-3-carbohydrazide (14)

Derivative 14 was obtained as a white solid in 72% yield from the condensation of 6 with benzaldehyde and recrystallization from ethanol. mp 216 °C; IR (cm^–1): 3495, 3365, 3320 (NH), 1670, 1635, 1600 (C=N), 1550, 1530, 720; ¹H NMR (300 MHz, DMSO-d₆): δ 6.30 (2H, br, NH₂), 7.25–8.11 (6H, m, Ar + =CH), 11.85 (1H, s, O=C–NH–N=); ¹³C NMR (DMSO-d₆): δ 116.3, 126.8, 126.9, 127.8, 127.9, 128.6, 128.7, 128.9, 129.3, 132.8, 136.7, 148.6, 155.3, 158.5, 180.8; MS (ESI) (m/z): [M + H]⁺ 335.1149. Anal. Calcd for C₁₈H₁₄N₄O₃ (334.32.): C, 64.66; H, 4.22; N, 16.76. Found: C, 64.78; H, 4.38; N, 16.61.

4-Amino-5-benzoyl-N′-[(1E)-(4-fluorophenyl)methylidene]-1,2-oxazole-3-carbohydrazide (15)

Derivative 15 was obtained as a white solid in 74% yield from the condensation of 6 with 4-fluorobenzaldehyde and recrystallization from dioxane. mp 237 °C; IR (cm^–1): 3410, 3320, 3200 (N–H), 1690, 1645 (C=O), 1530, 1500, 710; ¹H NMR (DMSO-d₆): δ 6.45 (2H, br, NH₂), 7.55–8.10 (9H, m, Ar), 8.54 (1H, s, =CH), 12.59 (1H, s, NH–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 112.2, 127.8, 128.2, 128.4, 128.5, 129.2, 129.31, 129.9, 131.0, 133.6, 134.3, 136.5, 138.8, 147.8, 150.6, 156.3, 180.8; MS (ESI) (m/z): [M + H]⁺ 353.1044. Anal. Calcd for C₁₈H₁₃FN₄O₃ (352.31): C, 61.36; H, 3.72; N, 15.90. Found: C, 61.06; H, 3.50; N, 16.23.

4-Amino-5-benzoyl-N′-[(1E)-(3-chlorophenyl)methylidene]-1,2-oxazole-3-carbohydrazide (16)

Derivative 16 was obtained as a pale-yellow solid in 67% yield from the condensation of 6 with 3-chlorobenzaldehyde followed by recrystallization from dioxane. mp 238 °C; IR (cm^–1): 3405, 3320, 3210 (NH), 1690, 1645 (C=O), 1530, 1500, 710; ¹H NMR (DMSO-d₆): δ 6.42 (2H, br, NH₂), 7.1–8.2 (10H, m, Ar + =CH), 11.98 (1H, s, O=C–NH); ¹³C NMR (DMSO-d₆): δ 111.9, 127.2, 127.3, 128.2, 128.3, 128.4, 129.2, 129.3, 131.1, 133.2, 138.6, 147.1, 151.0, 156.3, 159.4, 179.2; MS (ESI) (m/z): [M + H]⁺ 369.0749. Anal. Calcd for C₁₈H₁₃ClN₄O₃ (368.77): C, 58.62; H, 3.55; N, 15.19. Found: C, 58.48; H, 3.70; N, 15.50.

4-Amino-5-benzoyl-N′-[(1E)-(4-chlorophenyl)methylidene]-1,2-oxazole-3-carbohydrazide (17)

Derivative 17 was obtained as a pale-yellow solid in 67% yield from the condensation of 6 with 4-chlorobenzaldehyde following recrystallization from dioxane. mp 239 °C; IR (cm^–1): 3490, 3360, 3260 (N–H), 1695, 1650 (C=O), 1600 (C=N), 1555, 1500, 1280, 720; ¹H NMR (300 MHz, DMSO-d₆): δ 6.45 (2H, br, NH₂), 7.53–8.08 (9H, m, Ar), 8.52 (1H, s, =CH), 12.53 (1H, s, HN–C=O); ¹³C NMR (DMSO-d₆): δ 112.1, 126.9, 127.2, 128.1, 128.3, 128.4, 129.2, 129.4, 131.1, 133.4, 138.7, 147.3, 149.9, 156.3, 158.4, 180.0; MS (ESI) (m/z): [M + H]⁺ 369.0749. Anal. Calcd for C₁₈H₁₃ClN₄O₃ (368.77): C, 58.62; H, 3.55; N, 15.19. Found: C, 58.50; H, 3.24; N, 14.91.

4-Amino-5-benzoyl-N′-[(1E)-(4-methoxyphenyl)methylidene]-1,2-oxazole-3-carbohydrazide (18)

Derivative 18 was obtained as a white solid from the condensation of 6 with 4-methoxybenzaldehyde followed by recrystallization from dioxane. 81% yield; mp 236 °C; IR (cm^–1): 3480, 3360, 3260 (NH), 1685, 1640 (C=O), 1605 (C=N), 1250 (C–O–C), 720; ¹H NMR (DMSO-d₆): δ 3.81 (3H, s, CH₃), 6.43 (2H, s, CH₃), 7.01–8.09 (9H, m, Ar), 8.46 (1H, s, =CH), 12.3 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 55.3, 114.4, 126.3, 128.7, 128.8, 129.0, 129.1, 133.1, 136.0, 138.3, 147.4, 149.9, 155.5, 161.1, 180.3; MS (ESI) (m/z): [M + H]⁺ 365.1244. Anal. Calcd for C₁₉H₁₆N₄O₄ (364.35): C, 62.63; H, 4.43; N, 15.38. Found: C, 62.48; H, 4.23; N, 15.69.

General Procedure for the Synthesis of Isoxazolo[4,5-e][1,2,4]triazepine Derivatives 19–26

An equimolar amount of the appropriate aliphatic or aromatic aldehyde was added to a solution of compound 7 or 8 (0.01 mol) in 60 mL of anhydrous dioxane in the presence of a catalytic amount of p-toluene sulfonic acid. The reaction mixture was stirred for 8–10 h at 60 °C and poured then into cold water. After neutralization with 2% aqueous bicarbonate, the precipitate was separated by filtration, washed with 25 mL of methanol, and purified by column chromatography (CHCl₃/methanol, 9:1).

6-Acetyl-8-methyl-5-(propan-2-yl)-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (19)

Derivative 19 was obtained as a pale-yellow solid from the condensation of 7 with isobutyl aldehyde followed by recrystallization from dioxane. 49% yield; mp 189 °C; IR: 3490, 3360, 3180 (N–H), 1695, 1680, 1660 (C=O), 1615, 1600 (C=N), 1530, 1450, 1380, 1260, 700; ¹H NMR (300 MHz, DMSO-d₆): δ 1.04 (3H, d, CH₃), 1.07 (3H, d, CH₃), 1.80 (1H, m, CH), 2.13 (3H, s, CH₃), 2.31 (3H, s, CH₃), 7.08 (1H, m, N–CH=N), 8.04 (1H, s, NH–C=O), 8.09 (1H, s, NH–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 16.3, 19.1, 22.3, 25.7, 25.9, 71.0, 134.5, 147.8, 149.1, 160.7, 171.0; MS (ESI) (m/z): [M + H]⁺ 280.1415. Anal. Calcd for C₁₂H₁₇N₅O₃ (279.29): C, 51.60; H, 6.14; N, 25.08. Found: C, 51.24; H, 5.91; N, 24.82.

6-Acetyl-5-(4-chlorophenyl)-8-methyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-carboxamide (20)

Derivative 20 was obtained as a pale-yellow solid in 55% yield from the condensation of 7 with 4-chlorobenzaldehyde followed by recrystallization from dioxane. mp 216 °C; IR (cm^–1): 3495, 3345, 3190 (N–H), 1690, 1680 (C=O), 1620 (C=N), 1550, 1490, 715; ¹H NMR (300 MHz, DMSO-d₆): δ 2.13 (3H, s, −CH₃), 2.31 (3H, s, −CH₃), 6.98–7.01 (2H, d, Ar), 7.31–7.34 (2H, d, Ar), 7.39–7.41 (1H, d, J = 2 Hz, N–CH–N), 7.90–7.92 (1H, d, J = 2 Hz, =C–NH), 8.03 (1H, s, HN–C=O), 8.32 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 21.5, 25.6, 68.9, 127.6, 128.4, 128.9, 129.1, 129.2, 144.0, 147.6, 147.7, 161.7, 166.8; MS (ESI) (m/z): [M + H]⁺ 348.0858. Anal. Calcd for C₁₅H₁₄ClN₅O₃ (347.75): C, 51.81; H, 4.06; N, 20.14. Found: C, 51.71; H, 3.85; N, 19.89.

6-Acetyl-8-phenyl-5-(propan-2-yl)-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (21)

Derivative 21 was obtained as a pale-yellow solid in 55% yield from the condensation of 8 with isobutyl aldehyde followed by recrystallization from dioxane. mp 224 °C; IR (cm^–1): 3490, 3365, 3180 (N–H), 1695, 1680 (C=O), 1615, 1600 (C=N), 1530; ¹H NMR (300 MHz, DMSO-d₆): δ 1.02 (3H, d, −CH₃), 1.04 (3H, d, CH₃), 1.75–1.92 (1H, m, CH), 2.31 (3H, s, CH₃), 5.93–5.9 (1H, 2d, J = 2 Hz, N–CH–N), 7.44–7.54 (3H, m, Ar), 7.62–7.64 (1H, d, J = 2 Hz, =CNH), 7.71–7.74 (2H, d, Ar), 7.98 (1H, s, HN–C=O), 8.28 (1H, s, HN–C=O); ¹³C NMR: δ 16.3, 19.1, 22.3, 25.8, 71.0, 128.3, 128.9, 130.0, 133.5, 134.5, 144.3, 147.8, 149.1, 160.7, 171.0; MS (ESI) (m/z): [M + H]⁺ 342.1561. Anal. Calcd for C₁₇H₁₉N₅O₃ (341.36): C, 59.81; H, 5.61; N, 20.52. Found: C, 59.83; H, 5.64; N, 20.50.

6-Acetylo-5-(pentan-3-yl)-8-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (22)

Derivative 22 was obtained as a yellow solid in 49% yield from the condensation of 8 with ethylbutylaldehyde followed by recrystallization from tetrahydrofuran (THF). mp 222 °C; IR (cm^–1): 3490, 3370, 3190 (N–H), 1695, 1680 (C=O), 1615, 1600 (C=N), 1550, 710; ¹H NMR (300 MHz, DMSO-d₆): δ 0.68 (3H, t, CH₃), 0.78 (3H, t, CH₃), 1.28 (4H, m, 2 × CH₂), 1.51 (1H, m, CH), 2.27 (3H, s, CH₃), 6.15 (1H, dd, J = 2 Hz, N–CH–N), 7.49–7.52 (3H, m, Ar), 7.61–7.63 (1H, d, J = 2 Hz, =CH), 7.68–7.71 (2H, m, Ar), 7.95 (1H, s, HN–C=O), 8.25 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.0, 16.3, 19.2, 22.1, 22.2, 70.0, 128.4, 12.3, 128.9, 130.6, 133.7, 142.8, 146.5, 149.0, 159.6, 170.4; MS (ESI) (m/z): [M + H]⁺ 370.1873. Anal. Calcd for C₁₉H₂₃N₅O₃ (369.41): C, 61.77; H, 6.28; N, 18.96. Found: C, 62.02; H, 5.98; N, 19.21.

6-Acetyl-5-pentyl-8-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (23)

Derivative 23 was obtained as a pale-yellow solid in 59% yield from the condensation of 8 with hexanal followed by recrystallization from THF. mp 150 °C; IR (cm^–1): 3495, 3365, 3250 (N–H), 1696, 1680 (C=O), 1600 (C=N), 1550, 700; ¹H NMR (300 MHz, DMSO-d₆): δ 2.29 (3H, s, CH₃), 6.34 (1H, m, N–CH–N), 7.51–7.70 (5H, m, phenyl), 7.7–7.73 (1H, d, =CH), 7.98 (1H, s, HN–C=O), 8.27 (1H, s, N–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 13.6, 21.7, 22.3, 22.9, 25.0, 28.5, 30.3, 66.9, 128.3, 128.8, 128.9, 130.0, 133.7, 134.5, 144.1, 148.0, 149.6, 160.7, 170.7; MS (ESI) (m/z): [M + H]⁺ 370.1873. Anal. Calcd for C₁₉H₂₃N₅O₃ (369.41): C, 61.77; H, 6.28; N, 18.96. Found: C, 61.59; H, 6.33; N, 18.75.

6-Acetyl-5-undecyl-8-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (24)

Derivative 24 was obtained as a light-yellow solid in 71% yield from the condensation of 8 with lauryl aldehyde followed by recrystallization from THF. mp 188 °C; IR (cm^–1): 3490, 3360, 3200 (NH), 1690, 1680 (C=O), 1620, 1600 (C=N), 1540, 1370; ¹H NMR: δ 0.81 (3H, t, CH₃), 1.14 (18H, m, 9 × CH₂), 1.41 (2H, m, CH₂), 2.26 (3H, s, CH₃), 6.32 (1H, m, CH), 7.47–7.69 (5H, m, phenyl), 7.71 (1H, d, =CNH), 7.95 (1H, s, HN–CO), 8.25 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 13.9, 17.0, 22.0, 22.3, 23.0, 28.0, 28.5, 28.6, 28.8, 31.2, 64.9, 127.7, 128.3, 128.6, 128.8, 130.0, 134.6, 148.1, 149.5, 160.6, 169.6; MS (ESI) (m/z): [M + H]⁺ 454.2813. Anal. Calcd for C₂₅H₃₅N₅O₃ (453.57): C, 66.20; H, 7.78; N, 15.44. Found: C, 65.89; H, 15.21; N, 15.63.

6-Acetyl-5-(4-fluorophenyl)-8-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (25)

Derivative 25 was obtained as a light-yellow solid in 66% yield from the condensation of 8 with 4-fluorobenzaldehyde followed by recrystallization from dioxane. mp 222 °C; IR (cm^–1): 3490, 3390, 3250 (NH), 1700, 1690 (C=O), 1620, 1600 (C=N), 1510, 1350, 760; ¹H NMR: δ 2.41 (3H, s, CH₃), 7.08–7.62 (10H, m, 2 × phenyl + N–CH–N), 7.65 (1H, s, HN–C=O), 7.78–8.80 (1H, d, J = 2 Hz, =CNH), 8.31 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 22.8, 67.3, 116.0, 127.9, 128.5, 128.7, 128.9, 129.0, 130.3, 133.1, 134.5, 146.4, 148.6, 149.2, 160.2, 163.5, 172.2; MS (ESI) (m/z): [M + H]⁺ 394.1309. Anal. Calcd for C₂₀H₁₆FN₅O₃ (393.37): C, 61.07; H, 4.10; N, 17.80. Found: C, 60.87; H, 3.92; N, 17.99.

6-Acetyl-5-(4-chlorophenyl)-8-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carbamide (26)

Derivative 26 was obtained as a light-yellow solid in 72% yield from the condensation of 8 with 4-chlorobenzaldehyde followed by recrystallization from dioxane. mp 216 °C; IR (cm^–1): 3490, 3390, 3245, 3190 (NH), 1700, 1690 (C=O), 1620 (C=N), 1550, 1370, 700; ¹H NMR (300 MHz, DMSO-d₆): δ 2.41 (3H, s, CH₃), 7.08–7.63 (10H, m, 2 × phenyl + N–CH–N), 7.65 (1H, s, HN–C=O), 7.78–7.8 (1H, d, =CNH), 8.31 (1H, s, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 22.1, 66.9, 118.0, 127.8, 128.5, 128.4, 128.5, 128.9, 129.0, 130.0, 132.6, 136.1, 146.3, 149.0, 160.7, 172.0; MS (ESI) (m/z): [M + H]⁺ 410.1014 Anal. Calcd for C₂₀H₁₆ClN₅O₃ (409.82): C, 58.61; H, 3.94; N, 17.00. Found: C, 58.43; H, 4.13; N, 16.85.

General Procedure for the Synthesis of N′- and N″-Hydrazones 27–36 from Compounds 9(29,30)

To a solution compound 9 (10 mmol) in 120 mL of absolute ethanol was added an equimolar amount of the appropriate aliphatic or aromatic aldehyde. The reaction mixture was stirred for 6–8 h at 50–60 °C, and the volume was reduced to half by evaporation. The mixture was kept in a freezer overnight. The solid was separated by filtration, washed with 20 mL of methanol, and purified by recrystallization from suitable solvents.

4-(Acetamido)-5-{(1E)-1-[(2E)(2-methylpropylidene)hydrazinylidene]ethyl}-1,2-oxazole-3_NN-carboxamide (27)

Derivative 27 was obtained as a white solid in 73% yield from the condensation of 9 with isobutyl aldehyde followed by recrystallization from dioxane. mp 169 °C; IR (cm^–1): 3390, 3325, 3200 (N–H), 1690, 1660 (CO), 1615, 1600 (C=N), 1550, 1345; ¹H NMR (300 MHz, DMSO-d₆): δ 1.01 (3H, t, CH₃), 1.52 (2H, m, CH₂), 2.16 (3H, s, CH₃), 2.21 (3H, s, CH₃), 7.49 (1H, d, N=CH), 7.80 (1H, s, HN–CO), 8.11 (1H, s, HN–CO), 9.55 (1H, s, HN–CO); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.6, 14.7, 18.9, 19.1, 23.1, 117.1, 124.0, 129.4, 143.7, 144.8, 149.7, 153.2, 154.7, 156.2, 158.7, 159.6, 160.5, 162.8, 169.2; MS (ESI) (m/z): [M + H]⁺ 280.404. Anal. Calcd for C₁₂H₁₇N₅O₃ (279.29): C, 51.60; H, 6.14; N, 25.08. Found: C, 51.34; H, 5.93; N, 24.94.

4-Acetamido-5-{(1E)-1-[(2E)-(but-2-en-1-ylidene)hydrazinyliden]ethyl}-1,2-oxazole-3-carboxamide (28)

Derivative 28 was obtained as a white solid in 58% yield from the condensation of 9 with crotonaldehyde followed by recrystallization from ethanol. mp 196 °C; IR (cm^–1): 3390, 3320, 3200 (NH), 1690, 1660 (C=O), 1615, 1600 (C=N), 1515, 1320; ¹H NMR (300 MHz, DMSO-d₆): δ 1.92 (3H, d, CH₃), 1.99 (3H, s, CH₃), 2.30 (3H, s, CH₃), 6.30–6.58 (2H, m, CH=CH), 7.75 (1H, s, HN–CO), 8.10 (1H, d, N=CH), 8.13 (1H, s, HN–CO), 9.56 (1H, s, HN–CO); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.2, 18.6, 22.6, 116.6, 128.9, 144.3, 154.2, 155.7, 160.1, 162.3, 168.7; MS (ESI) (m/z): [M + H]⁺ 278.1248. Anal. Calcd: for C₁₂H₁₅N₅O₃ (277.27): C, 51.98; H, 5.45; N, 25.26. Found: C, 51.71; H, 5.19; N, 24.89.

4-(Acetamido)-5-{(1E)-1-[(2E)-(2-ethylbutylidene)hydrazinylidene]ethyl}-1,2-oxazole-3-carboxamide (29)

Derivative 29 was obtained as a white solid from the condensation of 9 with 2-ethylbutyraldehyde followed by recrystallization from ethanol. 48% yield; mp 168 °C; IR (cm^–1): 3390, 3320, 3200 (NH), 1690, 1660 (C=O), 1510, 1320; ¹H NMR (300 MHz, DMSO-d₆): δ 0.83 (3H, t, CH₃), 1.61 (4H, m, CH₂, CH₂), 1.93 (3H, t, CH₃), 2.09 (3H, s, CH₃), 2.29 (3H, s, CH₃), 7.75 (1H, s, HN–CO), 8.10 (1H, d, N=CH), 8.13 (1H, s, HN–CO), 9.55 (1H, HN–C=O); ¹³C NMR (300 MHz, DMSO-d₆): δ 8.2, 14.0, 14.8, 23.0, 30.7, 74.9, 115.2, 139.9, 156.2, 159.1, 160.6, 165.9, 167.0, 169.2; MS (ESI) (m/z): [M + H]⁺ 308.1717. Anal. Calcd for C₁₄H₂₁N₅O₃ (307.34): C, 54.71; H, 6.89; N, 22.79. Found: C, 54.40; H, 7.01; N, 22.95.

4-(Acetamido)-5-[(1E)-1-(2-hexylidenehydrazinylidene)ethyl]-1,2-oxazole-3-carboxamide (30)

Derivative 30 was obtained as a white solid from the condensation of 9 with n-hexanal followed by recrystallization from ethanol. 72% yield; mp 170 °C; IR (cm^–1): 3390, 3310, 3220 (NH), 1690, 1660 (C=O), 1635, 1600 (C=N), 1510, 1360; ¹H NMR (300 MHz, DMSO-d₆): δ 0.85 (3H, t, CH₃), 1.26–1.33 (6H, m), 1.9 (3H, s, CH₃), 2.25 (3H, s, CH₃), 2.30–2.40 (2H, d, CH₂), 7.5 (1H, s, =CH), 7.75 (1H, s, CONH), 8.11 (1H, s, CONH), 9.6 (1H, s, CONH); ¹³C NMR (300 MHz, DMSO-d₆): δ 8.2, 14.1, 14.8, 23.0, 30.6, 73.8, 115.3, 138.2, 155.9, 158.9, 160.1, 165.3, 166.1, 170.0; MS (ESI) (m/z): [M + H]⁺ 308.1717. Anal. Calcd for C₁₄H₂₁N₅O₃ (307.34): C, 54.71; H, 6.89; N, 22.79. Found: C, 54.99; H, 7.12; N, 23.08.

4-(Acetamido)-5-[(1E)-1-(2E)(phenylidenehydrazinylidene)ethyl]-1,2-oxazole-3-carboxamide (31)

Derivative 31 was obtained as a white solid from the condensation of 9 with benzaldehyde followed by recrystallization from dioxane. 60% yield; mp 234 °C; IR (cm^–1): 3390, 3310, 3200 (NH), 1695, 1660 (C=O), 1635, 1615 (C=N), 1500, 1290, 700; ¹H NMR (300 MHz, DMSO-d₆): δ 2.03 (3H, s, CH₃), 2.39 (3H, s, CH₃), 7.53–7.91 (6H, m, Ar + HN–CO), 8.12 (1H, s, HN–CO), 8.50 (1H, s, N + CH), 9.61 (1H, s, HN–CO); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.5, 22.6, 66.0, 116.8, 128.5, 128.9, 131.5, 133.7, 154.6, 155.7, 159.6, 160.1, 170.0; MS (ESI) (m/z): [M + H]⁺ 314.1238. Anal. Calcd for C₁₅H₁₅N₅O₃ (313.31): C, 57.50; H, 4.83; N, 22.35. Found: C, 57.26; H, 4.55; N, 22.05.

4-(Acetamido)-5-[(1E)-1-{(2E)-[(pyridin-4-yl)methylidene]hydrazinylidene}-ethyl]-1,2-oxazolo-3-carboxamide (32)

Derivative 32 was obtained as a white solid from the condensation of 9 with pyridine-4-carbonylaldehyde followed by recrystallization from dioxane. 87% yield; mp 244 °C; IR (cm^–1): 3380, 3340, 3240 (NH), 1690, 1670 (C=O), 1630, 1610 (C=N), 1530, 1450; ¹H NMR (300 MHz, DMSO-d₆): δ 2.03 (3H, s, CH₃), 2.36 (3H, s, CH₃), 7.75 (1H, s, HN–CO), 7.81 (2H, d, pyridine), 8.14 (1H, s, HN–CO), 8.42 (1H, s, N=CH), 8.74 (2H, d, pyridine), 9.64 (1H, s, HN–CO); ¹³C NMR (300 MHz, DMSO-d₆): δ 15.1, 23.1, 67.0, 117.6, 122.4, 141.0, 150.8, 150.9, 154.9, 156.1, 157.1, 158.3, 160.5, 169.3; MS (ESI) (m/z): [M + H]⁺ 315.1174. Anal. Calcd for C₁₄H₁₄N₆O₃ (314.30): C, 53.50; H, 4.49; N, 26.74. Found: C, 53.26; H, 4.22; N, 27.06.

4-(Acetamido)-5-[(1E)-1-{(2E)-[(4-fluorophenyl)methylidene]hydrazinylidene}ethyl]-1,2-oxazole-3-carboxamide (33)

Derivative 33 was obtained as a white solid from the condensation of 9 with 4-fluorobenzaldehyde followed by recrystallization from dioxane. 56% yield; mp 246 °C; IR (cm^–1): 3395, 3310, 3200 (NH), 1700, 1660 (C=O), 1620, 1600 (C=N), 1510, 1300; ¹H NMR (300 MHz, DMSO-d₆): δ 2.04 (3H, s, CH₃), 2.40 (3H, s, CH₃), 7.33–7.94 (5, Ar + HN–CO), 8.16 (1H, s, HN–CO), 8.53 (1H, s, N=CH), 9.65 (1H, s, HNCO); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.5, 22.6, 115.9, 116.2, 116.9, 130.4, 130.6, 130.7, 158.6, 163.0, 166.2, 169.2; MS (ESI) (m/z): [M + H]⁺ 332.1153. Anal. Calcd for C₁₅H₁₄FN₅O₃ (331.30): C, 54.38; H, 4.36; N, 21.14. Found: C, 54.49; H, 4.50; N, 20.92.

4-(Acetamido)-5-[(1E)-1-{(2E)-[(4-chlorophenyl)methylidene]hydrazinylidene}ethyl]-1,2-oxazole-3-carboxamide (34)

Derivative 34 was obtained as a pale-yellow solid from the condensation of 9 with 4-chlorobenzaldehyde followed by recrystallization from dioxane. 62% yield; mp 250 °C; IR (cm^–1): 3395, 3310, 3200 (N–H), 1695, 1660 (C=O), 1610, 1600 (C=O), 1520, 1300; ¹H NMR (300 MHz, DMSO-d₆): δ 2.93 (3H, s, CH₃), 2.38 (3H, s, CH₃), 7.57–7.93 (4H, m, Ar), 8.16 (1H, s, CONH), 8.52 (1H, s, N=CH), 9.65 (1H, s, CONH); ¹³C NMR (300 MHz, DMSO-d₆): δ 15.0, 23.4, 117.6, 129.5, 130.6, 133.1, 136.9, 156.4, 159.0, 160.9, 169.5; MS (ESI) (m/z): [M + H]⁺ 348.0858. Anal. Calcd for C₁₅H₁₄ClN₅O₃ (347.75): C, 51.81; H, 4.06; N, 20.14. Found: C, 51.62; H, 3.89; N, 19.90.

4-Acetamido-5-[(1E)-1-{(2E)-[(2-hydroxyphenyl)methylidene]hydrazinylidene}ethyl]-1,2-oxazole-3-carboxamide (35)

Derivative 35 was obtained as a white solid from the condensation of 9 with 2-hydroxybenzaldehyde followed by recrystallization from ethanol. 50% yield; mp 124 °C; IR (cm^–1): 3500, 3395, 3310, 3200 (N–H), 1695, 1660 (C=O), 1630, 1610 (C=N), 1510, 1320; ¹H NMR (300 MHz, DMSO-d₆): δ 2.04 (3H, s, CH₃), 2.25 (3H, s, CH₃), 6.94–7.79 (5H, m, Ar + HN–CO), 8.17 (1H, s, HN–CO), 8.87 (1H, s, N=CH), 9.67 (1H, s, HN–CO), 10.95 (1H, s, OH–phenol in DMSO); ¹³C NMR (300 MHz, DMSO-d₆): δ 15.9, 23.1, 117.0, 117.6, 119.0, 120.1, 131.0, 134.0, 156.0, 159.2, 160.6, 162.2, 169.3; MS (ESI) (m/z): [M + H]⁺ 330.1197. Anal. Calcd for C₁₅H₁₅N₅O₄ (329.31): C, 54.71; H, 4.59; N, 21.27. Found: C, 54.93; H, 4.85; N, 20.98.

4-(Acetamido)-5-[(1E)-1-{(2E)-[(4-trifluoromethylphenyl)methylidene]hydrazinylidene}ethyl]-1,2-oxazole-3-carboxamide (36)

Derivative 36 was obtained as a yellow solid in 68% yield from the condensation of 9 with 4-trifluoromethylbenzaldehyde followed by recrystallization from dioxane. mp 243 °C; IR (cm^–1): 3395, 3310, 3205 (NH), 1695, 1665 (C=O), 1640, 1620 (C=N), 1510, 1330; ¹H NMR (300 MHz, DMSO-d₆): δ 0.66 (3H, d, CH₃), 0.89 (3H, d, CH₃), 1.77 (1H, m, CH), 2.39 (3H, s, CH₃), 5.94 (1H, d, N=CH), 7.52–7.87 (6H, m, Ar + HN–CO), 8.11 (1H, s, HN–CO), 9.60 (1H, s, HN–CO); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.6, 22.7, 117.1, 125.5, 129.0, 129.3, 131.2, 137.5, 154.7, 155.7, 157.6, 160.1, 169.2; MS (ESI) (m/z): [M + H]⁺ 382.1122. Anal. Calcd for C₁₆H₁₄F₃N₅O₃ (381.30): C, 50.40; H, 3.70; N, 18.37. Found: C, 50.25; H, 3.95; N, 18.09.

General Procedure for the Preparation of 5-(1,2-Oxazol-3-yl)-2,3-dihydro-1,3,4-oxadiazole Derivatives 37–41

A solution of compound 12, 13, 15, 16, or 17 (0.005 mmol) in a mixture of acetic anhydride (20 mL) and anhydrous pyridine (20 mL) was stirred at room temperature for 15 h. The reaction mixture was poured into ice water, and the solid was collected by filtration, washed with cool water, and dried. The crude product was purified by column chromatography on silica gel and/or recrystallization from a suitable solvent.

1-[5-(4-Amino-5-benzoyl-1,2-oxazol-3-yl)-2-(propan-2-yl)-1,3,4-oxadiazol-3(2H)yl]ethan-1-one (37)

Derivative 37 was obtained from 12 as a white solid in 72% yield following recrystallization from ethanol. mp 151 °C; IR (cm^–1): 3490, 3380 (NH), 1695, 1660 (C=O), 1630, 1610 (C=N), 1550, 1515, 1380; ¹H NMR (300 MHz, DMSO-d₆): δ 0.84 (3H, d, CH₃), 1.01 (3H, d, CH₃), 2.28 (1H, m, CH), 2.30 (3H, s, CH₃), 6.24 (1H, s, O–CH–N), 6.28 (2H, br, NH₂), 7.57–8.09 (5H, m, phenyl); ¹³C NMR (300 MHz, DMSO-d₆): δ 18.7, 21.0, 21.3, 23.0, 92.6, 124.7, 128.8, 128.9, 129.0, 133.2, 135.8, 136.4, 146.8, 147.9, 157.0, 167.9, 180.3; MS (ESI) (m/z): [M + H]⁺ 343.1400. Anal. Calcd for C₁₇H₁₈N₄O₄ (342.34): C, 59.64; H, 4.45; N, 16.37. Found: C, 59.50; H, 4.62; N, 16.55.

1-[5-(4-Amino-5-benzoyl-1,2-oxazol-3-yl)-2-(pentan-3-yl)-1,3,4-oxadiazol-3(2H)yl]ethan-1-one (38)

Derivative 38 was obtained from 13 as a white solid, recrystallized from ethanol in 66% yield. mp 143 °C; IR (cm^–1): 3490, 3380 (NH), 1690, 1660 (C=O), 1630, 1615 (C=N), 1550, 1380; ¹H NMR (300 MHz, DMSO-d₆): δ 0.94 (3H, t, CH₃), 0.99 (3H, t, CH₃), 1.19 (1H, m, CH), 1.42–1.51 (4H, m, 2 × 2CH₂), 2.29 (3H, s, CH₃), 6.29 (2H, br, NH₂), 6.37 (1H, s, O–CH–N), 7.57–8.09 (5H, m, Ar); ¹³C NMR (300 MHz, DMSO-d₆): δ 11.1, 11.3, 18.7, 18.9, 21.0, 21.3, 94.9, 124.5, 128.8, 128.9, 129.0, 133.2, 135.8, 142.1, 147.9, 157.5, 166.9, 180.5; MS (ESI) (m/z): [M + H]⁺ 371.1714. Anal. Calcd for C₁₉H₂₂N₄O₄ (370.40): C, 61.61; H, 5.99; N, 15.13. Found: C, 61.41; H, 6.21; N, 14.89.

1-[5-(4-Amino-5-benzoyl-1,2-oxazol-3yl)-2-(3-chlorophenyl)-1,3,4-oxadiazol-3(2H)yl]ethan-1-one (39)

Derivative 39 was obtained from 16 as a pale-yellow solid in 72% yield following recrystallization from dioxane. mp 187 °C; IR (cm^–1): 3493, 3382 (NH), 1695, 1660 (C=O), 1630, 1610 (C=N), 1550, 1520; ¹H NMR (300 MHz, DMSO-d₆): δ 2.34 (3H, s, CH₃), 6.38 (2H, br, NH₂), 7.24 (1H, s, O–CH–N), 7.48–8.09 (9H, m, Ar); ¹³C NMR (300 MHz, DMSO-d₆): δ 21.7, 91.8, 126.1, 127.3, 129.3, 130.6, 131.3, 133.7, 133.9, 136.32, 138.4, 142.7, 147.3, 147.6, 168.1, 180.8; MS (ESI) (m/z): [M + H]⁺ 411.0855. Anal. Calcd for C₂₀H₁₅ClN₄O₄ (410.81): C, 58.47; H, 3.68; N, 13.64. Found: C, 58.69; H, 3.31; N, 13.87.

1-[5-(4-Amino-5-benzoyl-1,2-oxazol-3-yl)-2-(4-chlorophenyl)-1,3,4-oxadiazol-(2H)yl]ethan-1-one (40)

Derivative 40 was obtained from 17 as a pale-yellow solid in 70% yield following recrystallization from dioxane. mp 193 °C; IR (cm^–1): 3480, 3375, 3200 (NH), 1680, 1655 (C=O), 1620, 1610 (C=N); ¹H NMR (300 MHz, DMSO-d₆): δ 2.33 (3H, s, CH₃), 6.37 (2H, br, NH₂), 7.25 (1H, s, O–CH–N), 7.51–8.1 (9H, m, Ar); ¹³C NMR (300 MHz, DMSO-d₆): δ 21.3, 128.8, 128.9, 128.9, 133.2, 134.6, 135.8, 135.8, 142.2, 146.8, 147.1, 167.5, 180.3; MS (ESI) (m/z): [M + H]⁺ 411.0855. Anal. Calcd for C₂₀H₁₅ClN₄O₄ (411.81): C, 58.47; H, 3.68; N, 13.64. Found: C, 58.39; H, 3.55; N, 13.45.

1-[5-(4-Amino-5-benzoyl-1,2-oxazol-3yl)-2-(4-methoxyphenyl)-1,3,4-oxadiazol-(2H)yl]ethan-1-one (41)

Derivative 41 was obtained from 18 as a white solid in 65% yield following recrystallization from ethanol. mp 173 °C; IR (cm^–1): 3460, 3380 (NH), 1680, 1660 (C=O), 1620, 1615 (C=N), 1550, 1380, 1360; ¹H NMR: δ 2.33 (3H, s, CH₃), 3.76 (3H, s, O–CH₃), 6.37 (2H, br, NH₂), 6.97 (2H, d, Ar), 7.19 (1H, s, O–CH–N), 7.43–8.1 (7H, m, Ar); ¹³C NMR (300 MHz, DMSO-d₆): δ 20.9, 21.7, 56.1, 92.8, 114.6, 128.3, 128.8, 129.2, 129.3, 132.2, 133.7, 136.3, 137.0, 142.8, 147.3, 161.0, 167.7, 168.8, 180.8, 191.7; MS (ESI) (m/z): [M + H]⁺ 407.1350. Anal. Calcd for C₂₁H₁₈N₄O₅ (406.39): C, 62.06; H, 4.46; N, 13.79. Found: C, 61.86; H, 4.69; N, 13.50.

General Procedure for the Synthesis of Compounds 42–45

A solution of compound 30, 32, 33, or 36 (1 mmol) in a mixture of acetic anhydride (7 mL) and anhydrous pyridine (7 mL) was stirred at room temperature for 36 h. The excess acetic anhydride and pyridine were evaporated off under reduced pressure (1 mmHg), and the residual oil was triturated with anhydrous methanol. The obtained solid was separated by filtration, washed with absolute ethyl ether, and dried. The samples were purified by recrystallization from suitable solvents.

4,6-Diacetyl-8-methyl-5-phenyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (42)

Derivative 42 was obtained as a light-yellow solid from the cyclization of 31 followed by recrystallization from anhydrous dioxane and anhydrous THF to a constant. mp (179 °C). 65% yield; IR (cm^–1): 3450, 3350, 3200 (NH), 1705, 1700, 1690 (C=O), 1620, 1600 (C=N), 1540, 1240; ¹H NMR (300 MHz, DMSO-d₆): δ 2.30 (3H, s, CH₃), 2.34 (2H, s, CH₃), 2.46 (3H, s, CH₃), 7.50–7.91 (5H, m, Ar), 7.96 (1H, s, HN–CO), 8.32 (1H, s, HN–CO), 8.34 (1H, s, N–CH–N); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.7, 25.7, 25.72, 117.4, 128.7, 128.9, 131.8, 133.4, 154.6, 155.4, 159.1, 160.2, 162.6, 171.7; MS (ESI) (m/z): [M + H]⁺ 356.1346. Anal. Calcd for C₁₇H₁₇N₅O₄ (355.34): C, 57.46; H, 4.82; N, 19.71. Found: C, 57.45; H, 4.7.3; N, 19.60.

4,6-Diacetyl-5-(4-fluorophenyl)-8-methyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (43)

Derivative 43 was obtained as a yellow solid in 58% yield from the cyclization of compound 33 following recrystallization from anhydrous THF. mp 182 °C; IR (cm^–1): 3450, 3350, 3200 (NH), 1710, 1700, 1695 (C–O), 1620, 1600 (C=N), 1540, 1245; ¹H NMR (300 MHz, DMSO-d₆): δ 2.26 (3H, s, CH₃), 2.31 (3H, s, CH₃), 2.43 (3H, s, 3H₃), 7.31–7.97 (4H, m, Ar), 8.00 (1H, s, HN–CO), 8.30 (1H, s, HN–CO), 8.34 (1H, s, N–CH–N); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.8, 26.2, 26.2, 116.4, 116.7, 118.0, 131.6, 131.7, 155.4, 155.9, 159.5, 159.7, 163.0, 172.2; MS (ESI) (m/z): [M + H]⁺ 374.1252. Anal. Calcd for C₁₇H₁₆FN₅O₄ (373.33): C, 54.69; H, 4.32; N, 18.76. Found: C, 54.41; H, 4.53; N, 18.94.

4,6-Diacetyl-5-(4-chlorophenyl)-8-methyl-5,6-dihydro-4H-[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (44)

Derivative 44 was obtained as a yellow solid in 60% yield from the cyclization of compound 34 followed by recrystallization from anhydrous dioxane. mp 209 °C; IR (cm^–1): 3450, 3350, 3200 (NH), 1705, 1700, 1690 (C=O), 1540, 1240; ¹H NMR (300 MHz, DMSO-d₆): δ 2.29 (3H, s, CH₃), 2.32 (3H, s, CH₃), 2.39 (3H, s, CH₃), 7.56–7.94 (4H, m, Ar), 7.99 (1H, s, HN–CO), 8.33 (1H, s, HN–CO + −CO), 8.37 (1H, s, N–CH–N); ¹³C NMR (300 MHz, DMSO-d₆): δ 14.4, 25.7, 25.71, 117.5, 129.0, 130.1, 130.19, 130.3, 132.3, 136.4, 154.9, 155.4, 159.0, 159.1, 162.5, 171.1; MS (ESI) (m/z): [M + H]⁺ 390.0946. Anal. Calcd for C₁₇H₁₆ClN₅O₄ (389.79): C, 52.38; H, 4.14; N, 17.97. Found: C, 52.10; H, 4.30; N, 18.21.

4,6-Diacetyl-5-isopropyl-8-phenyl-5,6-dihydro-4H[1,2]oxazolo[4,5-e][1,2,4]triazepine-3-carboxamide (45)

Derivative 45 was obtained as a white solid 71% yield by the acetylation of compound 21 with acetyl chloride and anhydrous pyridine followed by recrystallization from anhydrous dioxane. mp 223 °C; IR (cm^–1): 3200 (CO–NH₂); ¹H NMR (300 MHz, DMSO-d₆): δ 0.78 (3H, s, CH₃), 1.01 (3H, d, CH₃), 1.74 (1H, m, CH), 2.36 (3H, s, CH₃), 2.38 (3H, s, CH₃), 6.78 (1H, s, N–CH–N), 7.51–7.73 (6H, m, 5H, Ar + 1HCONH), 8.21 (1H, s, CO–NH); ¹³C NMR (300 MHz, DMSO-d₆): δ 17.8, 18.1, 22.5, 25.5, 26.7, 70.1, 122.2, 128.7, 129.4, 130.3, 135.1, 141.3, 155.6, 160.3, 169.9, 173.6; MS (ESI) (m/z): [M + H]⁺ 384.1666. Anal. Calcd for C₁₉H₂₁N₅O₄ (383.40): C, 59.52; H, 5.52; N, 18.27. Found: C, 59.42; H, 5.53; N, 18.30.

6-Acetyl-N′-[(1E)-2-methylpropylidene]-8-phenyl-5-(propan-2-yl)-5,6-dihydro-H-[1,2]oxazolo-[4,5-e][1,2,4]triazepine-3-carbohydrazide (46)

Derivative 46 was obtained from the condensation of compound 12 (0.6 g, 2.0 mmol) with an equimolar amount of acetylhydrazine in absolute ethanol. The reaction was stirred at 60 °C for 8 h. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain the crude material, which was then diluted in hot anhydrous THF (20 mL), and 0.21 g (3.0 mmol) of fresh distilled isobutyl aldehyde and 1 mg of p-toluene sulfonic acid were added. The mixture was stirred at 60 °C 5 h, and the mixture was concentrated under reduced pressure; the residue was then diluted in hot anhydrous isopropanol. The mixture was kept in a freezer overnight. The solid was separated by filtration, washed with methanol, and purified by recrystallization from anhydrous acetone. 57% yield; mp 171 °C; IR (cm^–1): 3495, 3330, 3200 (NH), 1690, 1665 (C=O), 1550, 1280, 700; ¹H NMR (300 MHz, DMSO-d₆): δ 0.63–0.66 (3H, d, CH₃), 0.87–0.90 (3H, d, CH₃), 1.04 (3H, s, CH₃), 1.06 (3H, s, CH₃), 1.80 (1H, m, CH₃), 2.30 (3H, s, CH₃), 5.91–5.96 (1H, dd, C5H), 7.50–7.74 (6H, m, 5H, AR + 1N4H), 11.95 (1H, s, CONH); ¹³C NMR (300 MHz, DMSO-d₆): δ 16.3, 19.1, 19.4, 22.3, 25.7, 31.0, 66.9, 71.1, 128.3, 128.9, 130.0, 133.5, 134.6, 144.3, 154.9, 159.0, 171.1; MS (ESI) (m/z): [M + H]⁺ 411.2191. Anal. Calcd for C₂₁H₂₆N₆O₃ (410.46): C, 61.45; H, 6.38; N, 20.47. Found: C, 61.10; H, 6.05; N, 20.19.

Methods

Biological Assay of the Compounds’ Antiproliferative Activity (Data in Table 1)

Six human cancer cell lines were used to evaluate the antiproliferative activities of 21, 22, 24, 37, 38, 39, 40, 41, and 46. The cell lines used in this study were biphenotypic B myelomonocytic leukemia cells (MV4-11), human colon adenocarcinoma cell lines sensitive and resistant to doxorubicin (LoVo and LoVoDx, respectively), and lung cancer cells (A549). The MV4-11 and A549 cell lines were purchased from the American Type Culture Collection (ATCC Rockville, Maryland, USA); the MCF-7 cell line was purchased from the European Collection of Authenticated Cell Cultures; LoVo and LoVoDx were generously provided by Prof. E. Borowski (Technical University of Gdańsk, Poland). All the cell lines were maintained at the Institute of Immunology and Experimental Therapy (HIIET), Wrocław, Poland. Additionally, murine normal fibroblasts (Balb/3T3) purchased from ATCC were used as a control cell line.

Human leukemia cells (MV-4-11) were cultured in RPMI 1640 medium (HIIET, Wroclaw) containing 10% fetal bovine serum, 2 mM l-glutamine, and 1 mM sodium pyruvate (all from Sigma-Aldrich, Germany). Human colon adenocarcinoma (LoVo and LoVoDx) and lung cancer cell lines (A549) were cultured in a mixture of Opti-MEM and RPMI 1640 (1:1) medium (Opti-MEM from Gibco, RPMI 1640 from PAA, Austria) supplemented with 5% fetal bovine serum (PAA, Austria), 2 mM l-glutamine, 1 mM sodium pyruvate (Sigma-Aldrich, Germany), and 10 μg/100 mL doxorubicin for LoVo/DX (Sigma-Aldrich, Germany). The Balb/3T3 cell line was cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Warsaw, Poland) supplemented with 10% fetal bovine serum and 2 mM l-glutamine. MCF-7 was cultured in Eagle’s minimum essential medium (Thermo Fisher Scientific, Warsaw, Poland) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1% nonessential amino acids (Sigma-Aldrich, Poznan, Poland) and 80 μg/mL insulin (Sigma-Aldrich, Poznan, Poland). All culture media contained antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Polfa-Tarchomin, Poland). All cell lines were cultured during the entire experiment in a humidified atmosphere at 37 °C and 5% CO₂.

In Vitro Antiproliferative Assays

Twenty-four hours before adding the tested compounds, all the cell lines were seeded in 96-well plates (Sarstedt, Germany) in the appropriate media with 10⁴ cells per well (except A549, which was seeded 0.25 × 10⁴ cells per well). All cell lines were exposed to the appropriate test compound at four different concentrations (100 to 0.1 μg/mL) for 72 h. Cells were also exposed to the reference drugs, cisplatin (Ebewe, Austria) and doxorubicin (IBA, Poland). Additionally, all cell lines were exposed to DMSO (solvent used for the test compounds) (Sigma-Aldrich, Germany) at concentrations corresponding to those present in the dilutions of the test compounds. For adherent cells, a sulforhodamine B assay was performed, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed for leukemia cells.

Solution of Sulforhodamine (SRB)

After 72 h of incubation with the test compounds, cells were fixed in situ by gently adding 50 μL of cold 50% trichloroacetic acid (POCH, Poland) to each well, and the cells were incubated at 4 °C for 1 h. Then, the wells were washed four times with water, and 50 μL of 0.4% solution of sulforhodamine B (Sigma-Aldrich, Germany) in 1% acetic acid (POCH, Poland) was added to each well, and the plates were incubated at room temperature for 30 min. After incubation, the unbound dye was removed by washing the plates four times with 1% acetic acid, whereas the stain bound to the cells was solubilized with 10 mM Tris base (Sigma-Aldrich, Germany). The absorbance of each solution was read with a Synergy H4 system (BioTek Instruments, USA) at a wavelength of 540 nm. The entire washing procedure was performed with a BioTek EL-406 washing station.²⁹

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

The antiproliferative effects of the tested compounds on leukemia cells were measured by an MTT assay. Thus, 20 μL of MTT solution (Sigma-Aldrich, Germany) was added to each well, and the plates were left in a cell incubator for 4 h to allow the cells to metabolize yellow MTT to blue formazan. Then, 80 μL of a lysing mixture prepared with 225 mL of dimethylformamide with 67.5 g of sodium dodecyl sulfate (both from Sigma-Aldrich, Germany) and 275 mL of distilled water was added to each well. Plates were incubated for 24 h for the formazan crystals to be released from the cells and dissolved, and then, the absorbance of each well was read with a Synergy H4 system (BioTek 2 Instruments, USA) at 570 nm.³⁰

The results are presented as the mean IC₅₀ (concentration of the tested compound that inhibits cell proliferation by 50%) ± standard deviation. For each experiment, the IC₅₀ values were calculated using a Prolab-3 system based on Cheburator 0.4, Dmitry Nevozhay software.³¹ Compound attraction were tested in triplicate, and each experiment was repeated at least thrice independently. The results are summarized in the table.

X-ray Diffraction Studies

The X-ray diffraction data of compound 21 were collected at 100 K for a crystal of size 0.10 × 0.12 × 0.37 mm on an Xcalibur κ-axis diffractometer equipped with an Atlas charge-coupled device camera and an Oxford Cryosystems Cryostream 800 temperature controller. The data were collected using graphite-monochromated Mo Kα (λ = 0.71073 Å) radiation. Data reduction and analytical numeric absorption correction using a multifaceted crystal model were performed with CrysAlisPro software.¹⁹ The structure was solved by direct methods using the SHELXT program¹⁹ and refined by full-matrix least-squares calculations on F² with SHELXL software.¹⁹ Nonhydrogen atoms were refined with anisotropic displacement parameters. H atoms were found from difference Fourier maps and were refined isotropically. In the final refinement cycles, C/N-bound H atoms were repositioned in their calculated positions and refined using a riding model with C–H = 0.95 (phenyl ring), 0.98 (methyl group), 1.00 Å (methine group), N–H = 0.88 Å and with U_iso(H) = 1.5U_eq(C) for the methyl group and U_iso(H) = 1.2U_eq(C/N) for the remaining H atoms.

Crystal data for 6-acetyl-5-isopropyl-8-phenyl-5,6-dihydro-4H-isoxazolo[4,5-e][1,2,4]triazepine-3-carboxamide 21: C₁₇H₁₉N₅O₃, T = 100 K, M = 341.37, monoclinic, space group I2/a, a = 9.670(3) Å, b = 14.298(4) Å, c = 26.433(6) Å, β = 95.90(3)°, V = 3635.3(17) ų, Z = 8, D_c = 1.25 g cm^–3, μ = 0.09 mm^–1, T_min = 0.98, T_max = 0.99, F(000) = 1440, θ_min–θ_max 2.6–29.5°, 29,277 measured reflections (−13 ≤ h ≤ 12; −19 ≤ k ≤ 19; −35 ≤ l ≤ 35), 4681 independent and 3500 observed reflections with I > 2σ(I), R_int = 0.043, 229 parameters, R = 0.047, wR² = 0.126 for all reflections, goodness of fit = 1.04, Δρ_min = −0.25, Δρ_max = 0.29.

The crystallographic information file was deposited with the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/; deposition number CCDC 1900751).¹⁹

The authors declare no competing financial interest.