Synthesis, Spectroscopic Characterization, and In Vitro Antibacterial Evaluation of Novel Functionalized Sulfamidocarbonyloxyphosphonates

Abstract

Several new sulfamidocarbonyloxyphosphonates were prepared in two steps, namely carbamoylation and sulfamoylation, by using chlorosulfonyl isocyanate (CSI), α-hydroxyphosphonates, and various amino derivatives and related (primary or secondary amines, β-amino esters, and oxazolidin-2-ones). All structures were confirmed by ¹H, ¹³C, and ³¹P NMR spectroscopy, IR spectroscopy, and mass spectroscopy, as well as elemental analysis. Eight compounds were evaluated for their in vitro antibacterial activity against four reference bacteria including Gram-positive Staphylococcus aureus (ATCC 25923), and Gram-negative Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), in addition to three clinical strains of each studied bacterial species. Compounds 1a–7a and 1b showed significant antibacterial activity compared to sulfamethoxazole/trimethoprim, the reference drug used in this study.

1. Introduction

The synthesis and reactivity of sulfamides (sulfonyl analogues of ureas) have attracted much interest in the last decades [1]. A large number of sulfamide derivatives have been reported to show biological activities such as anti-mycobacterial, anticonvulsant, anti-hypoglycemic, anticancer, and enzyme inhibition (e.g., carbonic anhydrase I, HIV-1 protease, elastase, carboxypeptidase A) [2,3,4,5,6,7,8,9]. These important compounds have been synthesized by various routes, most of them using the reaction of a sulfonyl chloride with ammonia or primary and secondary amines [10]. Another approach utilizes the amide exchange of a sulfamide by heating with an amine [11]. In parallel, many synthetic efforts have also focused on sulfonamide derivatives that have shown great potency to inhibit important biological targets such as cox-2, carbonic anhydrase (e.g., isoenzymes I, II, VII, IX), and NaV1.7, or to block, for example, the Chlamydia fatty acid synthesis [12,13,14,15,16].

In addition, the extensive interest in the synthesis of bifunctional sulfonamide or sulfamide-phosphonate derivatives is due to their broad biological activities. In Figure 1, the structures of six bifunctional compounds are depicted. Biasone et al. [17] demonstrated that analogues of α-biphenylsulfonylamino 2-methylpropyl phosphonate 1 exhibit potency against several matrix metalloproteinases (MMPs). New sildenafil analogue 2 containing a phosphonate group in the 5′-sulfonamide moiety of the phenyl ring has shown promising in vitro PDE5 inhibitory activity [18]. Sulfonamide derivative 3 containing a single difluoromethylene phosphonate group has been discovered to be a potent inhibitor of protein tyrosine phosphatase PTP1B [19]. A series of phosphonate derivatives of mycophenolic acid 4 were described as anticancer, antiviral, and anti-inflammatory agents [20]. Compound 5 shows the highest insecticidal activity against plant pests [21]. It should be pointed out that to the best of our knowledge, it is the only example of compounds containing a sulfamidocarbonyloxyphosphonate moiety described in the literature. Finally, Winum et al. [22] reported the synthesis of sulfamide analogues of fotemustine 6 along with preliminary in vitro evaluation on two human melanoma cell lines.

Since the 1930’s, sulfamide and sulfonamide derivatives have had a special place in the anti-infectious strategies and their therapeutic application continues to be investigated, as illustrated by this recent work on the use of sulfonamide agents against Staphylococcus aureus (SA) of the CNS [23]. They demonstrated that sulfadiazine and sulfamethoxazole (SMX) (Figure 2) exhibited strong activity against bacteria. Fosfomycin (Figure 2) is another well-known antibacterial agent with a structure containing a phosphonate motif and may be prescribed alone or in combination (e.g., with vancomycin). Unfortunately, year after year, increased bacterial resistance to sulfonamides/sulfamides [24] to the combination sulfamethoxazole-trimethoprim (SMX-TMP) [25] and to fosfomycin [26] has limited their use. Moreover, the appearance of multidrug resistant Gram-positive bacteria, in particular methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), has become a major health problem [27]. So, new research on drug discovery needs to be intensively developed for designing new antibacterial agents.

In continuation of our interest in the preparation of sulfonamide and sulfamide derivatives [28,29,30,31], we decided to include both motifs, sulfamido and phosphonate, on each targeted compound and then to obtain new hybrids also containing an α-phenyl on the phosphonate methylene. For this preliminary study, we opted to select a set of various substituents -NR₂R₃ and -PO(OR₁)₂ in order to shape the first SAR trends in this series (Figure 3). To link these motifs, we chose a carbonyloxy-type spacer present in compounds 5 and 6 (Figure 1). The first-step reaction using chlorosulfonyl isocyanate and the corresponding α-hydroxyphosphonate-type intermediate allowed us to synthesize all N-chlorosulfonyl carbamate intermediates. In the second step, the key structural sequence, sulfamidocarbonyloxyphosphonate, was achieved directly from various amines. The antibacterial activity of eight phosphonate derivatives (1a–7a and 1b) was studied against representative reference strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853, as well as diverse clinical strains. Inhibition zones were performed by the disc diffusion method and the MIC values were determined by the dilution broth method [32]. The combination SMX-TMP, currently employed to treat bacterial infections, was used as the reference standard.

2. Results and Discussion

2.1. Chemistry

The synthetic route for the preparation of a novel series of sulfamidocarbonyloxyphosphonates 1a–8a is outlined in Scheme 1. The synthesis was carried out in two steps. First, carbamoylation under anhydrous conditions of commercial chlorosulfonyl isocyanate with the corresponding α-hydroxyphosphonate (R₁ = methyl or ethyl), easily prepared in a single step [33,34], quantitatively afforded the corresponding N-chlorosulfonyl carbamate intermediate. Reaction with various primary or secondary amines in the presence of triethylamine at 0 °C then gave the target compounds 1a–8a in excellent yields (92–99%) within 60–90 min (

Table 1. The physical data and yields for sulfamidocarbonyloxyphosphonates 1a–8a synthesized from primary and secondary amines.

Entry	-NR2R3	Target Molecule	Yield %	m.p. °C
1a			99	131–133
2a			98	137–139
3a			98	144–146
4a			96	136–138
5a			96	187–189
6a			94	153–155
7a			93	152–154
8a			97	151–153

).

To increase the scope of this reaction, we synthesized other sulfamidocarbonyloxyphosphonates using diverse (S)-amino acid esters (Scheme 2). The isolated yields of the products 1b–3b, obtained as a mixture of diastereoisomers (

Table 2. Physical data and yields for sulfamidocarbonyloxyphosphonates 1b–3b synthesized from amino acid esters.

Entry	R4	Target Molecule	Yield	m.p. °C
1b			91	118–120
2b			94	125–127
3b			84	116–118

), were in the range of 84–94% yield after 90 min of reaction.

These satisfactory and encouraging results have prompted us to develop a third subseries (Scheme 3,

Table 3. Physical data and yield for the sulfamidocarbonyloxyphosphonate 1c synthesized from oxazolidin-2-one.

Entry	Target Molecule	Yield %	m.p. °C
1c		92	123–125

), by using oxazolidin-2-one as a building block in order to synthesize new potential bioactive molecules. The new compound 1c was obtained with a very good yield (92%).

Spectrometric methods confirmed the structures of all the sulfamidocarbonyloxyphosphonates synthesized. Their physicochemical and analytical data are depicted in Table 1, Table 2 and Table 3. The FT-IR spectrum showed the characteristic signals of the three functions, namely the carbamate NH stretching at 3300–3250 cm⁻¹ and its C=O stretching at 1750–1730 cm⁻¹, the phosphonate group at 1255–1234 cm⁻¹, and the sulfamide group with its two signals at 1185–1118 cm⁻¹ and 1384–1356 cm⁻¹. The molecular peak [M + H]⁺ obtained by ESI-MS was always present and corresponded to each synthesized compound. NMR spectra were recorded using CDCl₃ as the solvent and are available in the supplementary material part. The ¹H spectrum always exhibited a dramatically deshielded doublet at 6 ppm corresponding to the COOCH(Ph)POOR proton with its expected coupling constant ²J_H-P frequently around 12–14 Hz. The two methoxy groups of the phosphonate appeared as two separate doublets (³J_H-P~10 Hz) at 3.5 and 3.7 ppm, while the NH of the carbamate appeared as a broad singlet at δ 8–11 ppm. The ¹³C spectrum was also characteristic due to the expected doublets related to the presence of the phosphorus (J_C-P couplings): (i) the methoxy of the phosphonates at 54 ppm (²J_C-P~7–8 Hz), and (ii) the aromatic ring (³J_C-P~6 Hz and ⁴J_C-P~1–3 Hz) [35,36]. The ¹³C chemical shifts are particular, as the carbonyl of the carbamate at 150 ppm (doublet with a ³J_C-P = 12 Hz coupling constant) and the greatly deshielded COOCH(Ph)POOR carbon at 70 ppm (doublet with a ¹J_C-P = 170 Hz coupling constant).

To determine the initial interest of these novel functionalized sulfamidocarbonyloxyphosphonates as antibacterial agents, we only selected eight derivatives (including seven from the first sub-series) for testing their potency against sixteen bacterial strains. This first biological study can confirm the interest to modulate such a scaffold.

2.2. In Vitro Antibacterial Evaluation of Sulfamidocarbonyloxyphosphonates

A total of twelve clinical strains of Gram-positive and Gram-negative bacteria and four control strains (S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603) were used to investigate the antibacterial activity. The eight tested sulfamidocarbonyloxyphosphonate derivatives (compounds 1a–7a and 1b) showed antibacterial activity with a varying degree of inhibitory effect on the growth of the bacterial strains (

Table 4. Diameters of the inhibition zone (DIZ) of sulfamidocarbonyloxyphosphonate derivatives 1a–7a, 1b, and SXT toward Gram-positive and Gram-negative bacteria.

	Molecules	Diameters of Inhibition Zone (DIZ) in mm a
Bacterial Strains		1a	2a	3a	4a	5a	6a	7a	1b	SXT
S. aureus ATCC 25923		15	16	14	12	15	12	13	13	22
S. aureus 1		16	15	14	R b	18	13	14	12	20
S. aureus 2		16	15	16	R	16	15	12	13	18
S. aureus 3		17	14	15	R	17	14	13	15	18
E. coli ATCC 25922		24	23	25	25	23	21	23	18	20
E. coli 1		17	22	18	20	20	20	19	18	18
E. coli 2		22	19	24	24	22	18	23	20	18
E. coli 3		20	22	23	22	19	18	20	17	20
P. aeruginosa ATCC 27853		26	20	18	23	19	19	20	18	17
P. aeruginosa 1		24	20	20	18	18	20	18	20	R
P. aeruginosa 2		26	19	20	20	20	19	18	17	20
P. aeruginosa 3		25	21	21	22	22	21	19	R	18
K. pneumoniae ATCC 700603		22	19	13	20	19	20	21	19	22
K. pneumoniae 1		22	21	18	25	15	18	19	15	R
K. pneumoniae 2		20	21	17	25	20	18	16	R	17
K. pneumoniae 3		20	18	18	24	19	19	22	21	R

^a All tests were performed in triplicate. ^b R: Resistant.

and

Table 5. Minimum inhibitory concentrations (MICs) of the sulfamidocarbonyloxyphosphonate derivatives 1a–7a and 1b toward Gram-positive and Gram-negative bacteria.

	Molecules	MIC (µg/mL) a
Bacterial Strains		1a	2a	3a	4a	5a	6a	7a	1b
S. aureus ATCC 25923		128	128	256	512	256	128	256	15
S. aureus 1		128	256	256	R b	64	128	256	15
S. aureus 2		64	128	128	R	128	64	128	18
S. aureus 3		64	128	128	R	128	128	128	15
E. coli ATCC 25922		1	2	2	0.5	2	2	8	18
E. coli 1		32	16	16	4	8	16	4	18
E. coli 2		4	16	2	0.5	4	16	8	20
E. coli 3		8	32	4	1	16	4	32	17
P. aeruginosa ATCC 27853		0.5	1	4	2	2	1	4	18
P. aeruginosa 1		0.5	2	2	4	2	2	2	20
P. aeruginosa 2		1	2	2	4	4	2	8	17
P. aeruginosa 3		0.5	6	1	2	2	4	4	R
K. pneumoniae ATCC 700603		4	32	256	16	128	128	32	19
K. pneumoniae 1		4	64	32	2	128	64	128	15
K. pneumoniae 2		8	16	16	2	32	128	128	R
K. pneumoniae 3		8	16	32	4	128	64	64	21

^a All tests were performed in triplicate and STX was used as the positive control (MIC = 25 µg/mL). ^b R: Resistant.

).

The disk diffusion is just a qualitative method to determine whether a particular bacterium is susceptible to the action of a specific antimicrobial agent. The presence or the absence of a clear region around the disk is an indication of the inhibition or lack of inhibition of the bacterial growth. Then, the size of the zone of inhibition indicates the degree of sensitivity of bacteria to an antimicrobial drug. We could use the terms “resistant, intermediate, and sensitive” to discuss the results obtained. As shown in Table 4, the diameters of the inhibition zone (DIZ) of the tested compounds against the bacteria strains ranged from 12–26 mm. The values obtained with the positive control sulfamethoxazole-trimethoprim (SXT) ranged between 17 and 22 mm for both clinical and control strains. Furthermore, some P. aeruginosa and K. pneumoniae strains (P. aeruginosa 1, K. pneumoniae 1 and 3) were resistant (R) towards SXT. It should be noted that P. aeruginosa is known to be a multidrug resistant bacteria due to its remarkable ability of acquiring mechanisms of resistance to some antimicrobial agents.

Tested sulfamidocarbonyloxyphosphonate derivatives were more active toward Gram-negative bacteria than Gram-positive ones. Compound 4a was inactive on all three clinical strains of S. aureus (only DIZ = 12 mm for S. aureus ATCC 25923). Compounds 2a, 3a, 6a, 7a, and 1b exerted intermediate activity on S. aureus (12 ≤ DIZ ≤ 16 mm). The best activities on S. aureus (DIZ > 16 mm, result reported as sensitive) were observed with compounds 1a (S. aureus 3) and 5a (S. aureus 1 and 3) with zone sizes of 17, 18, and 17 mm, respectively. Nevertheless, SXT seems to give better results, with zone sizes of between 18 and 22 mm. On E. coli strains, all tested compounds and STX gave results with inhibition zones between 17 and 25 mm. Among them, compounds 3a and 4a were the most active molecules against E. coli, with inhibition diameters of 25 mm for E. coli ATCC 25922 and 24 mm for E. coli 2. For P. aeruginosa strains, the inhibition zones were between 17 and 26 mm. Their susceptibility was really marked with compounds 1a, 4a, and 5a, with zone sizes between 18 and 26 mm. Compound 1a showed the best activity against strains P. aeruginosa ATCC 27853 and P. aeruginosa 2, with an inhibition diameter of 26 mm. SXT exhibited less activity against the four P. aeruginosa strains tested. For example, in the cases of P. aeruginosa ATCC 27853 and P. aeruginosa 2, the inhibition zones of STX were equal to 17 and 20 mm, respectively. Concerning K. Pneumoniae, all tested sulfamidocarbonyloxyphosphonate derivatives were globally active, with inhibition zones superior to 15 mm. The best activity was obtained with compound 4a, with inhibition zones of 24 and 25 mm for clinical strains.

After the evidence of in vitro antibacterial activity against the tested strains in the disk diffusion test, the Minimum Inhibitory Concentration (MIC) values were determined. As shown in Table 5, most derivatives exhibited low MIC values against the different strains of bacteria employed when compared with STX (MIC = 25 µg/mL). All the tested compounds showed the best MIC values against E. coli and P. aeruginosa strains, ranging between 0.5 and 32 µg/mL. In particular, compounds 1a, 3a, and 6a exerted the most intense activity, especially on P. aeruginosa, with MIC values ranging between 0.5 and 1 µg/mL for 1a and 1 and 4 µg/mL for 3a and 6a. As regards compound 4a, it was very active against E. coli strains, with MIC values between 0.5 and 4 µg/mL. For K. pneumoniae, the best results were obtained with compounds 1a and 4a, with MIC values in the range of 4 to 8 µg/mL and 2 to 16 µg/mL, respectively. Concerning S. aureus strains, all MIC values were superior to 64 µg/mL, except for compound 1b (15 ≤ MIC ≤ 18 µg/mL).

Overall, our results showed that the sulfamidocarbonyloxyphosphonates possessed a good concentration dependent antibacterial activity, especially against the tested Gram-negative bacteria at MIC values ranging between 0.5–32 µg/mL for compound 1a, and 0.5 and 16 µg/mL for compound 4a. Among the eight compounds tested, only compound 1b exerted antibacterial activity against S. aureus.

3. Materials and Methods

3.1. General Information

All chemicals and solvents were purchased from common commercial sources and were used as received without any further purification. All reactions were monitored by TLC on silica Merck 60 F254 percolated aluminum plates and were developed by spraying with ninhydrin solution. Column chromatography was performed with Merck silica gel (230–400 mesh). Proton nuclear magnetic resonance (¹H NMR) spectra were recorded on Bruker or Jeol spectrometers at 400 MHz. Chemical shifts are reported in δ units (ppm) with TMS as the reference (δ 0.00). All coupling constants (J) are reported in Hertz. Multiplicity is indicated by one or more of the following: b (broad), s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), and m (multiplet). The Carbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on Bruker (Reinstetten, Germany) or Jeol (JNM-ECS400 (Tokyo, Japan) spectrometers at 100.62 MHz. Chemical shifts are reported in δ units (ppm) and coupling constants (J) are reported in Hertz. Phosphorus nuclear magnetic resonance (³¹P NMR) spectra and Fluor (¹⁹F NMR) nuclear magnetic resonance spectra were recorded on a Bruker spectrometer at 161.98 MHz and 316.48 MHz, respectively. Infrared spectra were recorded on a Perkin Elmer 600 (Waltham, Massachusetts, USA) spectrometer. The Mass spectra were recorded on a shimadzu QP 1100 Ex mass spectrometer operating at an ionization potential of 70 eV. Elemental analysis was recorded on a EURO E.A. 3700 apparatus. All melting points were recorded on a Büchi B-545 (Taufkirchen, Germany) apparatus in open capillary tubes.

Ultrasound assisted reactions were carried out using a FUNGILAB ultrasonic bath (Barcelona, Spain) with a frequency of 40 kHz and a nominal power of 250 W. The reactions were carried out in an open glass tube (diameter: 25 mm; thickness: 1 mm; volume: 20 mL) at room temperature.

3.2. Typical Experimental Procedure for the Synthesis of Sulfamidocarbonyloxyphosphonates 1a–8a, 1b–3b, and 1c

α-Hydroxyphosphonates were synthesized in 94% overall yield starting from benzaldehyde and trialkylphosphites under ultrasound irradiation according to the procedure described in reference [34].

A solution of α-hydroxyphosphonate (1.1 equiv) in anhydrous CH₂Cl₂ (5 mL) was added dropwise to a stirring solution of chlorosulfonyl isocyanate (CSI) (1 equiv) in anhydrous CH₂Cl₂ (5 mL) at 0 °C over a period of 20 min. The resulting solution was transferred to a mixture of primary or secondary amine (1.1 equiv) or amino acid ester or oxazolidin-2-one in anhydrous CH₂Cl₂ (10 mL) in the presence of triethylamine (1.1–1.5 equiv). The reaction mixture was stirred at 0 °C for less than 1–2 h, and then neutralized by adding a solution of aqueous HCl 0.1 M to pH 7. The organic layer was extracted, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The pure products were crystallized in a mixture of diethyl ether/n-hexane (1.5:1) at 6 °C overnight. The pure sulfamidocarbonyloxyphosphonates were finally filtered and dried in excellent yields.

(Dimethoxyphosphoryl)(phenyl)methyl (N-benzylsulfamoyl)carbamate (1a). White powder, 99% yield, m.p. 131–133 °C, R_f = 0.43 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3365, 3298, 1733, 1481, 1364, 1249, 1170. ¹H-NMR (400 MHz, CDCl₃) δ: 3.57 (d, 3H, ³J_H-P = 10.4 Hz, CH₃-OP), 3.77 (d, 3H, ³J_H-P = 10.8 Hz, CH₃-OP), 4.11 (dd, 1H, J₁ = 13.6 Hz, J₂ = 5.4 Hz, CH-N), 4.23 (dd, 1H, J₁ = 14.0 Hz, J₂ = 5.6 Hz, CH-N), 5.61 (bs, 1H, NH-SO₂), 6.00 (d, 1H, ²J_H-P = 12.0 Hz, CH*-OP), 7.18–7.28 (m, 5H, H-Ar), 7.36–7.42 (m, 3H, H-Ar), 7.47–7.53 (m, 2H, H-Ar), 8.90 (bs, 1H, NH-C=O). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 48.15 (CH₂), 54.19 (d, J_C-P = 7 Hz, POCH₃), 54.48 (d, J_C-P = 7 Hz, POCH₃), 72.52 (d, J_C-P = 172 Hz, CH*-OP), 128.03 (2C, d, J_C-P = 6 Hz), 128.18 (2C), 128.34 (2C), 128.54, 128.96 (2C, d, J_C-P = 4 Hz), 129.58, 132.41, 135.54, 150.49 (d, J_C-P = 11 Hz, C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 19.10. Anal. Calc. for C₁₇H₂₁N₂O₇PS: C 47.66, H 4.94, N 6.54, S 7.48. Found: C 47.71, H 4.89, N 6.52, S 7.44%. ESI-MS: (m/z) = 429.1 [M + H]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl(N-(2-methoxyphenyl)sulfamoyl)carbamate (2a). White powder, 98% yield, m.p. 137–139 °C, R_f = 0.40 (CH₂Cl₂/MeOH, 90:10). IR(KBr, cm⁻¹): 3342, 3275, 1733, 1489, 1361, 1252, 1136. ¹H-NMR (400 MHz, CDCl₃) δ: 3.50 (d, 3H, ³J_H-P= 10.8 Hz, CH₃-OP), 3.54 (s, 3H, CH₃-O), 3.62 (d, 3H, ³J_H-P = 10.8 Hz, CH₃-OP), 5.94 (d, 1H, ²J_H-P = 14.0 Hz, CH*-OP), 6.75 (dd, 1H, J₁ = 8.0 Hz, J₂ = 1.2 Hz, H_ortho-Ar OMe), 6.84 (td, 1H, J₁ = 7.6 Hz, J₂ = 1.2 Hz, H_metha-Ar), 7.07 (td, 1H, J₁ = 6.8 Hz, J₂ = 1.2 Hz, H-Ar), 7.31–7.39 (m, 5H, H-Ar), 7.43 (dd, 1H, J₁ = 8.0 Hz, J₂ = 1.6 Hz, H_ortho-Ar NH), 7.55 (bs, 1H, NH-SO₂), 9.85 (bs, 1H, NH-C=O). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 54.16 (d, J_C-P = 7 Hz, POCH₃), 54.26 (d, J_C-P = 7 Hz, POCH₃), 55.79 (OCH₃), 72.20 (d, J_C-P = 174 Hz, CH*-OP), 111.09, 120.87, 121.04, 121.37, 125.95, 128.09 (2C, d, J_C-P = 6 Hz), 128.86, 129.33 (2C, d, J_C-P = 3 Hz), 132.52, 149.73, 150.09 (d, J_C-P = 12 Hz, C=O). ³¹P-NMR (161.98 CDCl₃) δ: 18.81. Anal. Calc. for C₁₇H₂₁N₂O₈PS: C 45.95, H 4.76, N 6.30, S 7.22. Found: C 45.90, H 4.81, N 6.28, S 7.26%. ESI-MS: (m/z) = 445.1 [M + H]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl(morpholinosulfonyl)carbamate (3a). White powder, 98% yield, m.p. 144–146 °C, R_f = 0.47 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3447, 3297, 1732, 1481, 1361, 1247, 1185, 769, 687. ¹H-NMR (400 MHz, CDCl₃) δ: 3.29–3.31 (m, 4H, 2 CH₂-N), 3.56 (d, 3H, ³J_H-P = 10.4 Hz, CH₃-OP), 3.65–3.67 (m, 4H, 2 CH₂-O), 3.84 (d, 3H,³J_H-P = 10.8 Hz, CH₃-OP), 6.02 (d, 1H, ²J_H-P = 13.6 Hz, CH*-OP), 7.37–7.40 (m, 3H, H-Ar), 7.51–7.55 (m, 2H, H-Ar), 9.92 (bs, 1H, NH-C=O). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 46.70 (2C, CH₂-N), 54.23 (d, J_C-P = 7 Hz, POCH₃), 54.38 (d, J_C-P = 7 Hz, POCH₃), 66.32 (2C, CH₂-O), 72.09 (d, J_C-P = 174 Hz, CH*-OP), 128.16 (2C, d, J_C-P = 6 Hz), 128.92 (2C, d, J_C-P = 1 Hz), 129.56 (d, J_C-P = 3 Hz), 132.46, 150.72 (d, J_C-P = 12 Hz, C=O). ³¹P-NMR (161.98 CDCl₃) δ: 18.93. Anal. Calc. for C₁₄H₂₁N₂O₈PS: C 41.18, H 5.18, N 6.86, S 7.85. Found: C 41.22, H 5.23, N 6.83, S 7.81%. ESI-MS: (m/z) = 409.1 [M + H]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl(N-(3-fluorophenyl)sulfamoyl)carbamate (4a). White powder, 96% yield, m.p. 136–138 °C, R_f = 0.41 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3311, 3297, 1758, 1477, 1355, 1251, 1166. ¹H-NMR (400 MHz, CDCl₃) δ: 3.62 (dd, 3H, J₁ = 38.8 Hz, J₂ = 10.4 Hz, CH₃-O), 3.72 (dd, 3H, J₁ = 10.4 Hz, J₂ = 1.2 Hz, CH₃-O), 5.95 (d, 1H, J = 13.6, CH*-O), 6.88–7.04 (m, 3H, H-Ar), 7.19–7.41 (m, 6H, H-Ar). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 54.85, 54.90, 71.86, 73.21, 128.14, 128.30, 129.23, 129.65, 12.91, 131.15, 131.75, 134.19, 134.56, 138.25, 138.45, 150.36. ³¹P-NMR (161.98 CDCl₃) 20.61. ¹⁹F-NMR (316.48 MHz, CDCl₃) δ: −111.62. Anal. Calc. for C₁₆H₁₈FN₂O₇PS: C 44.45, H 4.20, N 6.48, S 7.42. Found: C 44.40, H 4.23, N 6.52, S 7.41%. ESI-MS: (m/z) = 433.1 [M + H]⁺.

(Diethoxyphosphoryl)(phenyl)methyl(N-phenylsulfamoyl)carbamate (5a). White powder, 96% yield, m.p. 187–189 °C, R_f = 0.42 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3447, 3297, 1733, 1481, 1384, 1247, 1185. ¹H-NMR (400 MHz, CDCl₃) δ: 1.03 (t, 3H, J = 7.0 Hz, CH₃), 1.30 (t, 3H, J = 7.0 Hz, CH₃), 3.59–3.68 (m, 1H, CH₂-O), 3.82–3.90 (m, 1H, CH₂-O), 4.07–4.17 (m, 2H, CH₂-O), 5.82 (d, 1H, J = 8.8 Hz, CH*OP), 6.47 (s, 1H, NH-SO₂), 6.80 (dd, 2H, J₁ = 8.8 Hz, J₂ = 1.2 Hz, H-Ar), 7.02 (t, 1H, J = 7.6 Hz, H-Ar), 7.15 (t, 2H, J = 7.6 Hz, H-Ar), 7.20–7.26 (m, 5H, H-Ar).¹³C-NMR (100.62 MHz, CDCl₃) δ: 16.32 (CH₃), 16.59 (CH₃), 63.96 (CH₂), 64.10 (CH₂), 72.46 (d, J_C-P = 170 Hz, CH*-OP), 119.77 (2C), 124.46, 128.31 (2C, d, J_C-P = 6 Hz), 128.76 (2C), 128.90 (2C), 129.32, 134.25, 136.86, 150.40 (d, J_C-P = 16 Hz, C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 19.61. Anal. Calc. for C₁₈H₂₃N₂O₇PS: C 48.87, H 5.24, N 6.33, S 7.25. Found: C 48.93, H 5.21, N 6.28, S 7.26%. ESI-MS: (m/z) = 443.1 [M + H]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl(3,4-dihydroisoquinolin-2(1H)-yl)sulfonylcarbamate (6a). Color powder, 94% yield, m.p. 153–155 °C, R_f = 0.49 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3258, 1750, 1360, 1454, 1234, 1120. ¹H-NMR (400 MHz, CDCl₃) δ: 2.87 (t, 2H, J = 6.0 Hz, C_Ar-CH₂-CH₂), 3.55 (d, 3H, ³J_H-P = 10.0 Hz, CH₃-OP), 3.60 (t, 2H, J = 6.0 Hz, CH₂-CH₂-N), 3.75 (d, 3H, ³J_H-P = 10.0 Hz, CH₃-O), 4.52 (s, 2H, C_Ar-CH₂-N), 6.00 (d, 1H, ²J_H-P = 12.0 Hz, CH*-O), 7.01–7.08 (m, 2H, H-Ar), 7.14–7.16 (m, 2H, H-Ar), 7.33–7.36 (m, 3H, H-Ar), 7.48-7.51 (m, 2H, H-Ar), 9.91 (s, 1H, NH-C=O). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 28.38 (CH₂), 44.47 (NCH₂), 47.78 (NCH₂), 54.63 (2C, POCH₃), 72.04 (d, J_C-P = 178 Hz, CH*-OP), 126.4, 126.6, 127.1, 128.6 (2C), 128.8, 129.60 (2C), 129.80, 131.41, 132.4, 133.2, 150.95 (d, J_C-P = 16 Hz, C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 18.76. Anal. Calc. for C₁₉H₂₃N₂O₇PS: C 50.22, H 5.10, N 6.10, S 7.06. Found: C 50.19, H 5.15, N 6.16, S 7.10%. ESI-MS: (m/z) = 453.2 [M − H]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl(4-phenylpiperazin-1-yl)sulfonylcarbamate (7a). White powder, 93% yield, m.p. 152–154 °C, R_f = 0.50 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3337, 1741, 1449, 1360, 1248, 1167. ¹H-NMR (400 MHz, CDCl₃) δ: 3.10–3.40 (m, 4H, 2 CH₂-N-SO₂), 3.42–3.62 (m, 4H, 2 CH₂-N-C_Ar), 3.67 (d, 3H, J = 10.6 Hz, CH₃-O), 3.75 (d, 3H, J = 10.8 Hz, CH₃-O), 6.05 (d, 1H, ²J_H-P = 14.0 Hz, CH^*-O), 6.80–6.96 (m, 3H, H-Ar), 7.25–7.40 (m, 5H, H-Ar), 7.45–7.56 (m, 2H, H-Ar). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 46.78 (2C, NCH₂), 48.90 (2C, NCH₂), 54.61 (d, J_C-P = 7 Hz, POCH₃), 54.62 (d, J_C-P = 7 Hz, POCH₃), 70.86 (d, J_C-P = 142.4 Hz, CH*-OP), 117.37 (2C), 120.93, 127.87 (2C, d, J_C-P = 5 Hz), 128.57, 128.92 (2C), 129.65 (2C), 132.49, 136.81, 151.27 (d, J_C-P = 12 Hz, C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 19.82. Anal. Calc. for C₂₀H₂₆N₃O₇PS: C 49.68, H 5.42, N 8.69, S 6.63. Found: C 49.73, H 5.46, N 8.65, S 6.67%. ESI-MS: (m/z) = 482.3 [M − H]⁺.

(Diethoxyphosphoryl)(phenyl)methyl(N-propylsulfamoyl)carbamate (8a). White powder, 97% yield, m.p. 151–153 °C, R_f = 0.43 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3369, 3061, 1758, 1475, 1355, 1240, 1156, 763, 697. ¹H-NMR (400 MHz, CDCl₃) δ: 0.72 (t, 3H, J = 8.8 Hz, CH₃-Pr), 1.09 (t, 3H, J = 9.4 Hz, CH₃-OEt), 1.12–1.27 (m, 2H, CH₂-Pr), 1.37 (t, 3H, J = 9.4 Hz, CH₃-OEt), 2.48–2.59 (m, 1H, CH₂-N), 2.78–2.87 (m, 1H, CH₂-N), 3.67–4.05 (m, 2H, CH₂-OP), 4.25 (1H, m, NH), 4.74 (dq, 2H, ³J_H-P = 11.8 Hz, ³J_H-H = 7.5 Hz, CH₂-OP), 6.00 (dd, 1H, ²J_H-P = 11.3 Hz, J = 8.8 Hz, CH^*-O), 7.35–7.38 (m, 3H, H-Ar), 7.50–7.52 (m, 2H, H-Ar). Anal. Calc. for C₁₅H₂₅N₂O₇PS: C 44.11, H 6.17, N 6.86, S 7.85. Found: C 44.29, H 6.79, N 6.91, S 7.80%. ESI-MS: (m/z) = 409.2 [M + H]⁺.

(SR) and (SS)-Ethyl-2-((N-(((dimethoxyphosphoryl)(phenyl)methoxy)carbonyl)sulfamoyl)amino) -4-methylpentanoate (1b). White powder, 91% yield; m.p. 118–120 °C, R_f = 0.39 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3274, 1747 (l), 1470, 1371, 1251, 1164. ¹H-NMR (400 MHz, CDCl₃) δ: 0.81–0.87 (m, 12H, CH₃-CH_isop), 1.05–1.35 (m, 6H, O-CH₂-CH₃), 1.36–1.60 (m, 4H, 2CH_isop+ 1CH₂-CH_isop), 1.20 (m, 2H, 1CH₂-CH_isop), 3.51 (d, 3H, J = 10.6 Hz, CH₃-O), 3.52 (d, 3H, J = 10.6 Hz, CH₃-O), 3.60–3.75 (m, 1H, CH*-NH), 3.75–3.99 (m, 3H, -O-CH₂-CH₃ + CH*-NH), 3.78 (d, 6H, J = 10.8 Hz, CH₃-O), 4.00–4.25 (m, 2H, -O-CH₂-CH₃), 5.79 (bs, 1H, NH-SO₂), 5.96 (d, 1H, J = 13.9 Hz, CH^*-O), 6.00 (d, 1H, J = 14.3 Hz, CH^*-O), 6.21 (bs, 1H, NH-SO₂), 7.32–7.40 (m, 6H, H-Ar), 7.50-7.56 (m, 4H, H-Ar), 9.86 (bs, 1H, NH-C=O). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 14.04 (2CH₃), 22.76 (4C), 24.37 (2C), 41.95(2C), 54.19 (2POCH₃), 54.44 (2POCH₃), 55.55 (2C), 61.63 (2OCH₂), 72.13 (2C, d, J_C-P = 142.4 Hz, CH*-OP), 128.03 (4C), 128.12 (2C), 128.75 (4C, d, J_C-P = 5 Hz), 132.50 (2C), 150.60 (2C, d, J_C-P = 2 Hz, C=O), 172.01 (2C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 21.61. Anal. Calc. for C₁₈H₂₉N₂O₉PS: C 45.00, H 6.08, N 5.83, S 6.67. Found: C 45.07, H 6.04, N 5.81, S 6.72%. ESI-MS: (m/z) = 481.1 [M + H]⁺.

(SR) and (SS)-Ethyl-2-((N-(((dimethoxyphosphoryl)(phenyl)methoxy)carbonyl)sulfamoyl)amino) -3-phenylpropanoate (2b). White powder, 94% yield; m.p. 125–127 °C, R_f = 0.41 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3279, 1744 (l), 1455, 1373, 1249, 1162. ¹H-NMR (400 MHz, CDCl₃) δ: 1.01 (t, 3H, J = 7.6 Hz, CH₃-CH_2-O), 1.02 (t, 3H, J = 7.6 Hz, CH₃-CH_2-O), 2.85–3.15 (m, 4H, CH₂-Ar), 3.49 (d, 6H, J = 9.2 Hz, CH₃-O), 3.82 (d, 6H, J = 9.6 Hz, CH₃-O), 3.75–4.00 (m, 3H, CH*-NH + -O-CH₂-CH₃), 4.09–4.20 (m, 1H, CH*-NH), 4.25–4.50 (m, 3H, -O-CH₂-CH_{3 +} NH-SO₂), 4.86 (s, 1H, NH-SO₂), 5.97 (d, 1H, J = 13.5 Hz, CH^*-O), 5.98 (d, 1H, J = 14.3 Hz, CH^*-O), 7.00–7.12 (m, 2H, H-Ar), 7.11–7.41 (m, 14H, H-Ar), 7.42–7.48 (m, 4H, H-Ar). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 13.98 (2CH₃), 38.99 (2CH₂), 54.27 (2C, d, J_C-P = 6.9 Hz, POCH₃), 54.45 (2C, d, J_C-P = 6.9 Hz, POCH₃), 57.74 (2CH), 61.76 (2OCH₂), 71.14 (2C, d, J_C-P = 155.6 Hz, CH*-OP), 128.06 (4C, d, J_C-P = 6 Hz), 128.09 (4C), 128.57 (4C), 128.79 (4C), 129.51 (4C, d, J_C-P = 6 Hz), 132.2 (2C), 135.5 (2C), 150.69 (2C=O), 170.66 (2C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 23.42. Anal. Calc. for C₂₁H₂₇N₂O₉PS: C 49.02, H 5.29, N 5.44, S 6.23. Found: C 45.07, H 6.04, N 5.81, S 6.72%. ESI-MS: (m/z) = 515.21 [M + H]⁺.

(SR) and (SS)-Ethyl-2-((N-(((dimethoxyphosphoryl)(phenyl)methoxy)carbonyl)sulfamoyl) amino)-3-(1H-indol-3-yl) propanoate (3b). White powder, 84% yield; m.p. 116–118 °C; R_f = 0.39 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3274, 1747, 1471, 1371, 1250, 1164. ¹H-NMR (400 MHz, CDCl₃) δ: 0.99 (t, 3H, J = 7.20 Hz, CH₃-CH_2-O), 1.06 (t, 3H, J = 7.2 Hz, CH₃-CH_2-O), 3.11 (d, 4H, J = 6.0 Hz, CH₂-CH*), 3.30–3.50 (m, 8H, 2CH₃-O + 2CH*CO), 3.82–4.00 (m, 8H, 2CH₃-O + OCH₂), 4.21–4.26 (m, 2H, OCH₂), 6.20 (d, 2H, J = 7.8 Hz, CH^*-O), 6.68–6.98 (m, 6H, H-Ar), 7.18–7.26 (m, 8H, H-Ar), 7.37–7.40 (m, 6H, H-Ar), 9.60 (bs, 2H, NH-C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 20.61. Anal. Calc. for C₂₃H₂₈N₃O₉PS: C 49.91, H 5.10, N 7.59, S 5.79. Found: C 49.97, H 5.04, N 7.68, S 5.83%. ESI-MS: (m/z) = 553.21 [M]⁺.

(Dimethoxyphosphoryl)(phenyl)methyl ((2-oxooxazolidin-3-yl)sulfonyl)carbamate (1c). White powder; 92% yield; m.p. 123–125 °C; R_f = 0.38 (CH₂Cl₂/MeOH, 90:10). IR (KBr, cm⁻¹): 3255, 1748, 1663, 1357, 1254, 1118, 757, 629; ¹H-NMR (400 MHz, CDCl₃) δ: 3.40–3.43 (m, 2H, CH₂-N), 3.61 (d, 3H, ³J_H-P = 8.0 Hz, CH₃-OP), 3.75 (d, 3H, ³J_H-P = 8.0 Hz, CH₃-OP), 4.60–4.63 (m, 2H, CH₂-O), 6.04 (d, 1H, ²J_H-P = 12.0 Hz, CH*-OP), 7.31–7.35 (m, 3H, H-Ar), 7.37-7.39 (m, 2H, H-Ar). ¹³C-NMR (100.62 MHz, CDCl₃) δ: 46.58, 54.92 (d, J_C-P = 7 Hz, POCH₃), 54.95 (d, J_C-P = 7 Hz, POCH₃), 70.76, 71.02 (d, J_C-P = 171 Hz, CH*-OP), 127.93 (2C, d, J_C-P = 3 Hz), 128.75, 128.99 (2C, d, J_C-P = 2 Hz), 133.52, 155.06 (C=O), 155.12 (d, J_C-P = 12 Hz, C=O). ³¹P-NMR (161.98 MHz, CDCl₃) δ: 18.93. Anal. Calc. for C₁₃H₁₇N₂O₉PS: C 38.24, H 4.20, N 6.86, S 7.85. Found: C 38.20, H 4.25, N 6.89, S 7.81%. ESI-MS: (m/z) = 431.5 [M + Na]⁺.

3.3. Determination of In Vitro Antibacterial Activity

The antimicrobial activity of the synthesized compounds was evaluated in vitro against Gram positive and Gram negative bacteria. Serial dilutions of the tested compounds in acetone were made in a concentration range from 0.5 to 512 µg/mL. All tests were performed in triplicate.

Firstly, compounds 1a–7a and 1b were screened for antibacterial activity by using the Kirby Bauer disc diffusion test on Mueller-Hinton agar plates. The medium was poured into Petri plates and allowed to solidify. These plates were inoculated with a bacterial inoculum prepared in physiologically sterile water with an OD of about 0.08. Sterilized disks of 6 mm (Schleicher and Schule, Germany) were each impregnated with 20 µL of different concentrations of the compounds and were deposited on the plates. The latter were then left at room temperature for 2 h and incubated at 37 °C for 24 h. The diameters of the inhibition zones (mm) were measured in accordance with the recommendations of the clinical and laboratory standards institute (CLSI 2017) [32]. For each bacterial strain, the best inhibition zone obtained was reported in Table 4.

Secondly, the MIC values were determined by the dilution broth method following the procedure recommended by the CLSI [32]. The serial dilutions of compounds, ranging from concentrations of 0.5 to 512 μg/mL, T were inoculated with fresh bacterial inoculums and then incubated at 37 °C for 24 h. The MIC value was considered as the lowest concentration showing visual inhibition of growth. Sulfamethoxazole-trimethoprime (Bio-Rad, Marseille, France) was used as the positive control (CMI = 25 µg/mL). Disks embedded with acetone were used as a negative one.

4. Conclusions

In summary, 12 new and original sulfamidocarbonyloxyphosphonates were synthesized and fully characterized by ¹H, ¹³C, and ³¹P NMR spectroscopy, IR spectroscopy, and mass spectroscopy, as well as elemental analysis. The synthesized compounds 1a–7a and 1b were screened for in vitro evaluation as a proof of concept for designing new antibacterial agents containing both sulfamido and phosphonate moieties. Standard strains were chosen according to the screening protocol including Gram-positive and Gram-negative bacteria, which represent micro-organisms associated with important infections. All compounds showed promising in vitro antibacterial activity. Additionally, it has been demonstrated that our derivatives have more antibacterial effects on Gram-negative bacteria than Gram-positive ones except for compound 1b (R₁=CH₃, R₂=CH(iBu)COOEt, R₃=H). This latter is the only one active on both Gram-negative and Gram-positive bacteria. Compound 1a (R₁=CH₃, R₂=Bn, R₃=H) had more pronounced activity against P. aeruginosa, whereas compound 4a (R₁=CH₃, R₂=3-F-C₆H₄, R₃=H) had more activity on E. coli. Antibacterial effects will be investigated in further studies to explain the susceptibility of bacteria to our compounds. Further pharmacomodulation efforts are in progress to explore the impact of new substituents on the phenyl moiety and thereby will offer new expectations for sulfamidocarbonyloxyphosphonates as novel antibacterial agents.