# Synthesis, Characterization, X-Ray Crystal Structure, and Antimicrobial Activity  of 1,1′-(3,4-Diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone

Authors: Yahia Nasser Mabkhot; Fahad D. Aldawsari; S. S. Al-Showiman; Assem Barakat; M. Iqbal Choudhary; Sammer Yousuf
Journal: Journal of Chemistry
 (2014)

---

## Abstract

Synthesis of 1,1′-(3,4-diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone (4) is reported here. The structure of compound 4 was deduced by 1H-NMR, 13C-NMR, FT-IR, MS, microanalysis, and single-crystal X-ray diffraction. Compound crystallizes in the monoclinic space group P21/n with a = 9.3126(7) Å, b = 9.5867(7) Å, c = 20.2811(15) Å, α = 90°, β = 95.436(2)°, γ = 90°, V = 1802.5(2) Å3, and Z = 4. The molecules are packed in crystal structure by weak intermolecular C10–H10A⋯ S1 hydrogen bonding interactions. Compound 4 can be a useful intermediate for the synthesis of diphenylthieno[2,3-b]thiophene. Compound 4 was found to be active against Gram-positive bacteria (Bacillus subtilis and Staphylococcus pneumoniae) and Gram-negative bacteria (Escherichia coli) and also was found to be active against fungi (Aspergillus fumigatus and Candida albicans).


---

## Body

## 1. Introduction

Thienothiophenes skeletons are important in pharmaceutical research because of their versatile biological activities, such as antitumor, antiviral antibacterial, anticancer, antioxidant andβ-glucuronidase and α-glucosidase inhibition, antiglaucoma activity, and inhibitors of platelet aggregation properties [1–5]. Because of this thieno[2,3-b]thiophenes have been the focus of active research in recent years. We have the synthesis of certainbis-heterocycles containing thieno[2,3-b]thiophene derivatives. The molecules that were prepared were found to be potent α-glucosidase inhibitors (IC50 = 14.1
±
0.28
μM) with manyfold more activity than the standard acarbose (IC50 = 841
±
1.73
μM). Indeed, another example incorporating thieno[2,3-b]thiophene core was found to be a potent β-glucuronidase inhibition (IC50 = 0.003
±
0.09
μM) several hundred fold more active than standard D-saccharic acid 1,4-lactone (IC50 = 45.75
±
2.16
μM) [6, 7].The skeleton is identified as valuable scaffold for new heterocyclic compounds [8–11]. The structure of 1,1′-(3,4-diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone (4) was unambiguously deduced by single-crystal X-ray diffraction technique. Compound4 was also screened forin vitro antimicrobial activity.


## 2. Experimental

### 2.1. General

All the chemicals were purchased from various suppliers, including Sigma-Aldrich and Fluka, and were used without further purification, unless otherwise stated. All melting points were measured on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR Spectrum was recorded as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. The NMR spectra were recorded on a Varian Mercury Jeol-400 NMR spectrometer.1H-NMR (400 MHz) and 13C-NMR (100 MHz) were run in deuterated dimethyl sulphoxide (DMSO-d
6). Chemical shifts (δ) are referred to inppm whileJ-coupling constants were represented inHz. Mass spectra were recorded on a Jeol of JMS-600 H. Elemental analysis was carried out on Elmer 2400 Elemental Analyzer, CHN mode. The single-crystal X-ray diffraction measurements were performed using Bruker SMART APEXII CCD diffractometer.


### 2.2. Preparation of 1,1′-(3,4-Diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone

Method A. A mixture of 1,3-diphenylpropane-1,3-dione (1) (22.4 g, 0.1 mol) and anhydrous potassium carbonate (25 g) in DMF (30–40 mL) was stirred vigorously at room temperature for 5 min; then carbon disulfide (7.6 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting reaction mixture was cooled in ice bath, and then chloroacetone (18.5 mL, 0.2 mol) was added with continued stirring for 15 min. Then cooling bath was subsequently removed and the mixture was stirred for further 30 min. The solid product was collected by filtration and washed with water and dried crude product.Method B. A mixture of 1,3-diphenylpropane-1,3-dione (1) (22.4 g, 0.1 mol) and NaOEt (2 mol Na in 30–40 mL absolute EtOH) was stirred vigorously at room temperature for 5 min, and then carbon disulfide (7.6 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting reaction mixture was cooled in ice bath, and then chloroacetone (18.5 mL, 0.2 mol) was added with continued stirring for 15 min; then cooling bath was subsequently removed and the mixture was stirred for further 30 min. The solid product was collected by filtration and washed with water and dried [12, 13].Yield: 75%; m.p. 265°C; IR (ν
max
⁡): 1637 (C=O) cm−1; 1H-NMR (400 MHz, DMSO-d
6): δ 1.74 (s, 6H, CH3), 7.00–7.04 (m, 10H, Ph); 13C-NMR (100 MHz, DMSO-d
6): δ 31.37 (COCH3), 191.3 (C=O), 129.23, 129.55, 129.87, 134.79, 138.82, 141.84, 147.68 (Ar–C); MSm/z (%): 376 [M+, 60%], 300 (100), 226 (37), 184 (14); anal. calcd. for C22H16O2S2: C, 70.18; H, 4.28; S, 17.03; found: C, 70.07; H, 4.44; S, 17.11.


### 2.3. Crystal Structure Determination

Slow evaporation of glacial acetic acid solution of pure compound4 yielded colorless crystals. A crystal of dimensions 0.54 × 0.53 × 0.30 mm was selected for X-ray diffraction analysis. Data were collected on a Bruker APEX-II CCD area diffractometer and equipped with graphite monochromatic Mo Kα radiation (λ
=
71073 Å) at 293 (2) °K. Cell refinement and data reduction were carried out by Bruker SAINT [14]. SHELXS-97 [15, 16] was used to solve structure (Table 1). The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F
2. All the hydrogen atoms were placed in calculated positions. The crystal structure4 (Figure 1) was finally refined with R factor of 4.25% for 3362 unique reflections. Molecules were found to be packed in crystal lattice through intermolecular hydrogen bonding (Table 2).Table 1
The crystal and experimental data of compound4.
Empirical formula
C22H16O2S2
Formula weight
376.47
Temperature (K)
297(2)
MoK
α radiations, λ
0.71073 Å
Crystal system
Monoclinic
Space group
P21
/
n
a
9.3126(7) Å
b
9.5867(7) Å
c
20.2811(15) Å
β
95.436(2)°
Volume
1802.5(2)Å
3
Z
4
Calculated density
1.387 mg/m3
Absorption coefficient
0.309 mm−1
F(000)
784
Crystal shape and color
Colorless, block
Crystal size
0.54 × 0.53 × 0.30 mm
θ range
2.02 to 25.50°
h
/
k
/
l
−11,11/−11,11/−24,24
Reflections collected
10407
Reflections unique
3362
(R
int
⁡)
0.0261
R
1 with I > 2σ(I)
0.0360
R
2 with I > 2σ(I)
0.0925
R
1 for all data
0.0425
R
2 for all data
0.0980
Goodness of fit
1.042
max/minρ  e
Å
-
3
0.193/−0.176Table 2
Hydrogen bonding data for compound4.
D
H
A
D–H
H⋯A
D⋯A
D–H⋯A
C10
H10A
S1a
0.9300
2.8500
3.7542(2)
161.00
Symmetry codes:a
-
1
/
2
+
x, 1
/
2
-
y, -
1
/
2
+
z.Figure 1
The ORTEP diagram of the final X-ray model of compound4with displacement ellipsoids drawn at 30% probability level. H-atoms were placed and not included in refinement.


### 2.4. Antifungal Activity

Tested sample was screenedin vitro for its antifungal activity against various fungi, namely,Aspergillus fumigatus (RCMB 002568) andCandida albicans (RCMB 05036). The antifungal activity was performed by agar well diffusion method.Fungal strains were grown in 5 mL sabouraud dextrose broth (glucose : peptone; 40 : 10) for 3-4 days to obtain 105 CFU/mL cells. The fungal culture (0.1 mL) was spread uniformly on the sabouraud dextrose agar plates by sterilized triangular folded glass rod. Plates were left for 5–10 min so the culture is properly adsorbed on the surface. Now small wells of size 4 mm × 2 mm were cut into the plates with the help of well cutter and bottom of the wells was sealed with 0.8% soft agar to prevent the flow of test sample at the bottom of the well. 100μL of the tested samples (10 mg/mL) was loaded into the wells of the plates. Compound4 dissolved in DMSO, while pure DMSO was also used as control. The plates were kept for incubation at 30°C for 3-4 days and then examined for the formation of zones of inhibition. The test was performed three times for each fungus. Amphotericin B was used as standard antifungal drug.


### 2.5. Antibacterial Activity

Antibacterial activities were investigated by using agar well diffusion method, against theStaphylococcus pneumonia (RCMB 010010) andBacillus subtilis (RCMB 010067) {as Gram-positive bacteria} andPseudomonas aeruginosa (RCMB 010043) andEscherichia coli (RCMB 0100052) {as Gram-negative bacteria}. The solution of 5 mg/mL of compound in DMSO was prepared for testing against bacteria. Centrifuged pellets of bacteria from 24 h old culture containing approximately 104–106 CFU (colony forming unit) per mL were spread on the surface of nutrient agar (typetone 1%, yeast extract 0.5%, NaCl 0.5%, agar, and 1000 mL of distilled water, pH 7.0) which was autoclaved under 121°C for at least 20 min. Wells were created in medium with the help of sterile metallic bores and then cooled down to 45°C. The activity was determined by measuring the diameter of the inhibition zone (in mm). 100 μL of the tested samples (10 mg/mL) was loaded into the wells of the plates. Solution of compound was prepared in DMSO while DMSO was also loaded as control. The plates were kept for incubation at 37°C for 24 h and then the plates were examined for the formation of zone of inhibition. Each inhibition zone was measured three times by caliper to get an average value. The test was performed three times for each bacterium. Ampicillin and gentamicin were used as antibacterial standard drugs [17].


## 2.1. General

All the chemicals were purchased from various suppliers, including Sigma-Aldrich and Fluka, and were used without further purification, unless otherwise stated. All melting points were measured on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR Spectrum was recorded as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. The NMR spectra were recorded on a Varian Mercury Jeol-400 NMR spectrometer.1H-NMR (400 MHz) and 13C-NMR (100 MHz) were run in deuterated dimethyl sulphoxide (DMSO-d
6). Chemical shifts (δ) are referred to inppm whileJ-coupling constants were represented inHz. Mass spectra were recorded on a Jeol of JMS-600 H. Elemental analysis was carried out on Elmer 2400 Elemental Analyzer, CHN mode. The single-crystal X-ray diffraction measurements were performed using Bruker SMART APEXII CCD diffractometer.


## 2.2. Preparation of 1,1′-(3,4-Diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone

Method A. A mixture of 1,3-diphenylpropane-1,3-dione (1) (22.4 g, 0.1 mol) and anhydrous potassium carbonate (25 g) in DMF (30–40 mL) was stirred vigorously at room temperature for 5 min; then carbon disulfide (7.6 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting reaction mixture was cooled in ice bath, and then chloroacetone (18.5 mL, 0.2 mol) was added with continued stirring for 15 min. Then cooling bath was subsequently removed and the mixture was stirred for further 30 min. The solid product was collected by filtration and washed with water and dried crude product.Method B. A mixture of 1,3-diphenylpropane-1,3-dione (1) (22.4 g, 0.1 mol) and NaOEt (2 mol Na in 30–40 mL absolute EtOH) was stirred vigorously at room temperature for 5 min, and then carbon disulfide (7.6 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting reaction mixture was cooled in ice bath, and then chloroacetone (18.5 mL, 0.2 mol) was added with continued stirring for 15 min; then cooling bath was subsequently removed and the mixture was stirred for further 30 min. The solid product was collected by filtration and washed with water and dried [12, 13].Yield: 75%; m.p. 265°C; IR (ν
max
⁡): 1637 (C=O) cm−1; 1H-NMR (400 MHz, DMSO-d
6): δ 1.74 (s, 6H, CH3), 7.00–7.04 (m, 10H, Ph); 13C-NMR (100 MHz, DMSO-d
6): δ 31.37 (COCH3), 191.3 (C=O), 129.23, 129.55, 129.87, 134.79, 138.82, 141.84, 147.68 (Ar–C); MSm/z (%): 376 [M+, 60%], 300 (100), 226 (37), 184 (14); anal. calcd. for C22H16O2S2: C, 70.18; H, 4.28; S, 17.03; found: C, 70.07; H, 4.44; S, 17.11.


## 2.3. Crystal Structure Determination

Slow evaporation of glacial acetic acid solution of pure compound4 yielded colorless crystals. A crystal of dimensions 0.54 × 0.53 × 0.30 mm was selected for X-ray diffraction analysis. Data were collected on a Bruker APEX-II CCD area diffractometer and equipped with graphite monochromatic Mo Kα radiation (λ
=
71073 Å) at 293 (2) °K. Cell refinement and data reduction were carried out by Bruker SAINT [14]. SHELXS-97 [15, 16] was used to solve structure (Table 1). The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F
2. All the hydrogen atoms were placed in calculated positions. The crystal structure4 (Figure 1) was finally refined with R factor of 4.25% for 3362 unique reflections. Molecules were found to be packed in crystal lattice through intermolecular hydrogen bonding (Table 2).Table 1
The crystal and experimental data of compound4.
Empirical formula
C22H16O2S2
Formula weight
376.47
Temperature (K)
297(2)
MoK
α radiations, λ
0.71073 Å
Crystal system
Monoclinic
Space group
P21
/
n
a
9.3126(7) Å
b
9.5867(7) Å
c
20.2811(15) Å
β
95.436(2)°
Volume
1802.5(2)Å
3
Z
4
Calculated density
1.387 mg/m3
Absorption coefficient
0.309 mm−1
F(000)
784
Crystal shape and color
Colorless, block
Crystal size
0.54 × 0.53 × 0.30 mm
θ range
2.02 to 25.50°
h
/
k
/
l
−11,11/−11,11/−24,24
Reflections collected
10407
Reflections unique
3362
(R
int
⁡)
0.0261
R
1 with I > 2σ(I)
0.0360
R
2 with I > 2σ(I)
0.0925
R
1 for all data
0.0425
R
2 for all data
0.0980
Goodness of fit
1.042
max/minρ  e
Å
-
3
0.193/−0.176Table 2
Hydrogen bonding data for compound4.
D
H
A
D–H
H⋯A
D⋯A
D–H⋯A
C10
H10A
S1a
0.9300
2.8500
3.7542(2)
161.00
Symmetry codes:a
-
1
/
2
+
x, 1
/
2
-
y, -
1
/
2
+
z.Figure 1
The ORTEP diagram of the final X-ray model of compound4with displacement ellipsoids drawn at 30% probability level. H-atoms were placed and not included in refinement.


## 2.4. Antifungal Activity

Tested sample was screenedin vitro for its antifungal activity against various fungi, namely,Aspergillus fumigatus (RCMB 002568) andCandida albicans (RCMB 05036). The antifungal activity was performed by agar well diffusion method.Fungal strains were grown in 5 mL sabouraud dextrose broth (glucose : peptone; 40 : 10) for 3-4 days to obtain 105 CFU/mL cells. The fungal culture (0.1 mL) was spread uniformly on the sabouraud dextrose agar plates by sterilized triangular folded glass rod. Plates were left for 5–10 min so the culture is properly adsorbed on the surface. Now small wells of size 4 mm × 2 mm were cut into the plates with the help of well cutter and bottom of the wells was sealed with 0.8% soft agar to prevent the flow of test sample at the bottom of the well. 100μL of the tested samples (10 mg/mL) was loaded into the wells of the plates. Compound4 dissolved in DMSO, while pure DMSO was also used as control. The plates were kept for incubation at 30°C for 3-4 days and then examined for the formation of zones of inhibition. The test was performed three times for each fungus. Amphotericin B was used as standard antifungal drug.


## 2.5. Antibacterial Activity

Antibacterial activities were investigated by using agar well diffusion method, against theStaphylococcus pneumonia (RCMB 010010) andBacillus subtilis (RCMB 010067) {as Gram-positive bacteria} andPseudomonas aeruginosa (RCMB 010043) andEscherichia coli (RCMB 0100052) {as Gram-negative bacteria}. The solution of 5 mg/mL of compound in DMSO was prepared for testing against bacteria. Centrifuged pellets of bacteria from 24 h old culture containing approximately 104–106 CFU (colony forming unit) per mL were spread on the surface of nutrient agar (typetone 1%, yeast extract 0.5%, NaCl 0.5%, agar, and 1000 mL of distilled water, pH 7.0) which was autoclaved under 121°C for at least 20 min. Wells were created in medium with the help of sterile metallic bores and then cooled down to 45°C. The activity was determined by measuring the diameter of the inhibition zone (in mm). 100 μL of the tested samples (10 mg/mL) was loaded into the wells of the plates. Solution of compound was prepared in DMSO while DMSO was also loaded as control. The plates were kept for incubation at 37°C for 24 h and then the plates were examined for the formation of zone of inhibition. Each inhibition zone was measured three times by caliper to get an average value. The test was performed three times for each bacterium. Ampicillin and gentamicin were used as antibacterial standard drugs [17].


## 3. Results and Discussion

### 3.1. Title Compound4 Was Synthesized as Depicted in Scheme 1, in 75% Yield

Scheme 1
Synthetic pathway towards title compound4.The structure was deduced by combined use of IR,1H-NMR, 13C-NMR, and mass spectral data. Accordingly, the assigned structure was unambiguously establishedvia single-crystal X-ray diffraction.


### 3.2. Crystal Structure of Compound4

The asymmetric unit contains two molecules. The crystal structure of compound4 is composed of two planner thiophene rings (S1/C1-C3/C6 and S2/C3-C5/C6), fused along C3 and C6 plane having two phenyl (C7-C12/C13-C18) rings and ethanone (O1/C19-C20/O2/C21-C22) moieties attached to C1, C2, C4, and C5 atoms (Figure 1). Two thiophene (S1/C1-C3/C6 and S2/C3-C5/C6) and phenyl (C7-C12 and C13-C18) rings are each planner with maximum deviation of 0.011(2) Å for C2 and C6 atoms from the root mean square plane. The crystal molecules are linkedvia C10–H10A⋯S1 interaction to form chains arranged in azigzag fashion along the c-axis (Figure 2).Figure 2
The crystal packing of compound4. Dashed line indicates the intermolecular interactions. The hydrogen atoms not involved in intermolecular interactions are omitted for clarity.


### 3.3. Antimicrobial Activity of Compound4

Compound4 was evaluated against pathogenic microorganisms representing Gram-positive bacteria (Bacillus subtilis andStaphylococcus pneumoniae), Gram-negative bacteria (Pseudomonas aeruginosa andEscherichia coli), and fungi (Aspergillus fumigatus andCandida albicans), and the activities were compared with standard antibacterial and antifungal standard drug, specified in US pharmacopeia at 25 μg/mL. Compound4showed a relatively moderate inhibitory effect against Gram-positive bacteria (Bacillus subtilis andStaphylococcus pneumoniae) as compared to standard drug ampicillin. It also showed a relatively moderate inhibitory effect against Gram-negative bacteria (Escherichia coli), as comparable to that of the standard drug gentamicin. No activity was observed againstPseudomonas aeruginosa. Finally, compound4 was also found to be active against fungi (Aspergillus fumigatus andCandida albicans), compared to the standard drug amphotericin B. The results obtained are summarized in Table 3.Table 3
Antimicrobial activity of compound4.
Comp. number
Gram-positive bacteria
Gram-negative bacteria
Fungi
Staphylococcus
pneumoniae
Bacillus  subtilis
Pseudomonas  aeruginosa
Escherichia  coli
Aspergillus  fumigatus
Candida
albicans
4
12.9 ±  0.63
13.2  ±  0.58
NA
10.8 ± 0.44
18.7 ± 0.36
16.9 ±  0.27
Ampicillin
23.8  ±  0.2
32.4  ±  0.3
—
—
—
—
Gentamicin
—
—
17.3 ±  0.1
19.9  ±  0.3
—
—
Amphotericin B
—
—
—
—
23.7  ±  0.1
25.4 ±  0.1
Inhibition zones (mm).
NA: no activity.


## 3.1. Title Compound4 Was Synthesized as Depicted in Scheme 1, in 75% Yield

Scheme 1
Synthetic pathway towards title compound4.The structure was deduced by combined use of IR,1H-NMR, 13C-NMR, and mass spectral data. Accordingly, the assigned structure was unambiguously establishedvia single-crystal X-ray diffraction.


## 3.2. Crystal Structure of Compound4

The asymmetric unit contains two molecules. The crystal structure of compound4 is composed of two planner thiophene rings (S1/C1-C3/C6 and S2/C3-C5/C6), fused along C3 and C6 plane having two phenyl (C7-C12/C13-C18) rings and ethanone (O1/C19-C20/O2/C21-C22) moieties attached to C1, C2, C4, and C5 atoms (Figure 1). Two thiophene (S1/C1-C3/C6 and S2/C3-C5/C6) and phenyl (C7-C12 and C13-C18) rings are each planner with maximum deviation of 0.011(2) Å for C2 and C6 atoms from the root mean square plane. The crystal molecules are linkedvia C10–H10A⋯S1 interaction to form chains arranged in azigzag fashion along the c-axis (Figure 2).Figure 2
The crystal packing of compound4. Dashed line indicates the intermolecular interactions. The hydrogen atoms not involved in intermolecular interactions are omitted for clarity.


## 3.3. Antimicrobial Activity of Compound4

Compound4 was evaluated against pathogenic microorganisms representing Gram-positive bacteria (Bacillus subtilis andStaphylococcus pneumoniae), Gram-negative bacteria (Pseudomonas aeruginosa andEscherichia coli), and fungi (Aspergillus fumigatus andCandida albicans), and the activities were compared with standard antibacterial and antifungal standard drug, specified in US pharmacopeia at 25 μg/mL. Compound4showed a relatively moderate inhibitory effect against Gram-positive bacteria (Bacillus subtilis andStaphylococcus pneumoniae) as compared to standard drug ampicillin. It also showed a relatively moderate inhibitory effect against Gram-negative bacteria (Escherichia coli), as comparable to that of the standard drug gentamicin. No activity was observed againstPseudomonas aeruginosa. Finally, compound4 was also found to be active against fungi (Aspergillus fumigatus andCandida albicans), compared to the standard drug amphotericin B. The results obtained are summarized in Table 3.Table 3
Antimicrobial activity of compound4.
Comp. number
Gram-positive bacteria
Gram-negative bacteria
Fungi
Staphylococcus
pneumoniae
Bacillus  subtilis
Pseudomonas  aeruginosa
Escherichia  coli
Aspergillus  fumigatus
Candida
albicans
4
12.9 ±  0.63
13.2  ±  0.58
NA
10.8 ± 0.44
18.7 ± 0.36
16.9 ±  0.27
Ampicillin
23.8  ±  0.2
32.4  ±  0.3
—
—
—
—
Gentamicin
—
—
17.3 ±  0.1
19.9  ±  0.3
—
—
Amphotericin B
—
—
—
—
23.7  ±  0.1
25.4 ±  0.1
Inhibition zones (mm).
NA: no activity.


## 4. Conclusion

The synthesis and characterization of a new 1,1′-(3,4-diphenylthieno[2,3-b]thiophene-2,5-diyl)diethanone (4) were successfully achieved in high yield. The structure of4was confirmed finally by single-crystal X-ray diffraction. Compound4showed activity effect against Gram-negative bacteria (Bacillus subtilis andStaphylococcus pneumoniae), Gram-positive bacteria (Escherichia coli), and antifungal activity against (Aspergillus fumigatus andCandida albicans). The pivotal compound4 can be used as a synthon for new drugs with above cited.



---

*Source: 504860-2014-05-12.xml*