*2.8. Immuno-Detection of Native FhSOD1 and FhSOD3 in NEJ, Adult Parasites and Their Extracts*

Localisation of FhSOD1 and FhSOD3 in NEJ was conducted as follows: *F. hepatica* metacercariae (Italian isolate; Ridgeway Research, St. Briavels, UK) were excysted and cultured in RPMI 1640 medium containing 2 mM L-glutamine, 30 mM HEPES, 0.1% (*w*/*v*) glucose, 2.5 μg/mL gentamycin, and 10% foetal calf serum (ThermoFisher Scientific) for 24 h, as previously described [29]. NEJ were then fixed with 4% paraformaldehyde (PFA) in 0.1 M PBS (Sigma-Aldrich) pH 7.4, for 1 h at RT. After three washes in antibody diluent (PBS containing 0.1% (*v*/*v*) Triton X-100, 0.1% (*w*/*v*) bovine serum albumin and 0.1% (*w*/*v*) sodium azide; AbD buffer), the NEJ were incubated in rabbit pre-immune sera, anti-rFhSOD1, anti-rFhSOD3, or anti-rFhCL3 polyclonal antibodies (used as a non-related positive control) diluted 1:500 in AbD buffer overnight at 4 ◦C. After three washes in AbD, the NEJ were incubated in a 1:200 dilution of the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit IgG (Sigma-Aldrich) overnight at 4 ◦C in the dark. To counter-stain muscle tissue, the samples were incubated in AbD containing phalloidin-tetramethylrhodamine isothiocyanate (TRITC) (200 μg/mL) overnight in the dark at 4 ◦C. The NEJ were then whole-mounted onto slides using 10% glycerol solution containing 0.1 M propyl gallate and visualised under an Olympus Fluoview 3000 laser scanning confocal microscope using a PL APO CS 6 × 0 oil objective lens. Olympus type F immersion oil was used in viewing and all images were taken at room temperature.

Localisation of FhSOD1 and FhSOD3 in adult *F. hepatica* was conducted on parasites collected from the livers of infected sheep during post-mortem surveillance in Roscommon, Ireland. After collection, the parasites were washed in 1 × PBS and fixed in 4% PFA for 4 h at RT. After 4 h, the PFA was removed, replaced with 1 × PBS followed by an incubation for 1 h at RT. This washing process was repeated twice before dehydration in ascending ethanol and subsequent infiltration with JB-4 resin (Sigma-Aldrich EM0100). Serial 0.5 μM sections were mounted onto slides and probed with rabbit pre-immune sera, anti-rFhSOD1, anti-rFhSOD3, or anti-rFhCL1 polyclonal antibodies (used as a non-related positive control) diluted 1:1000 in PBST for 5 h at RT in a humid container. The sections were washed three times in PBST before addition of a 1:1000 dilution of the secondary antibody, FITC-labelled goat-anti-rabbit IgG (Sigma-Aldrich) in PBST, after which they were placed in a humid container and allowed to incubate overnight in the dark at 4 ◦C. The slides were once again washed three times in PBST and dried, and cover slips were mounted using 10% glycerol solution containing 0.1 M propyl gallate. The sections were visualised under a Leica DM2500 LED optical fluorescent microscope (Leica Microsystems, Wetzlar, Germany).

Adult *F. hepatica* E/S and somatic extract (10 μg/lane) were resolved by gel electrophoresis in 4–20% SDS-PAGE gels and electro-transferred onto nitrocellulose membranes prior to incubation in blocking buffer for 1 h at RT. The membranes were probed overnight at 4 ◦C with rabbit pre-immune sera, anti-rFhSOD1, or anti-rFhSOD3 at a 1:1000 dilution. After washing, the membranes were further probed with the secondary antibody alkaline phosphatase conjugated goat-anti-rabbit IgG at a 1:10,000 dilution for 1 h at RT. Following

final washes, the immune-reactive bands were visualised using the substrate SigmaFast BCIP/NBT (Sigma-Aldrich).


**8. Conclusion**

There is not a superior technique that can be recommended as the only "gold standard." For this reason, the surgical treatment of the rectocele must be individualized in each case according to the needs of the patient and surgeon's experience.

A transvaginal approach with posterior colporrhaphy and native tissue is recommended in cases of women with ODS who require surgical treatment, reporting the rate of possible post-surgical dyspareunia.

In cases with multicompartmental prolapse or with a very high rectocele, an abdominal approach is more suitable (**Figure 5**).

Non-absorbable mesh should not be used in the vaginal approach due to potential adverse effects.

An improvement in the results with the use of biological materials has not been demonstrated; however, their use increases the surgical costs.

Site-specific repair has a higher recurrence rate and is not recommended as the first technique of choice in patients with constipation.

Transanal approach for the treatment of posterior rectocele is associated with lower resolution of ODS symptoms and a higher recurrence rate with higher infection rates. This approach should be avoided in patients with fecal incontinence or known sphincteric damage, cases in which the association of perineoplasty or levatorplasty may be more indicated.

#### **Figure 5.**

*Option of rectocele treatment algorithm, ODS: Obstructive defecation syndrome, TV: Transvaginal, FI: Fecal incontinence, LVR: Laparoscopic ventral rectopexy, STARR: Stapled transanal rectal resection.*


*2.2. Syntheses*

#### 2.2.1. 3,3-,5,5--Tetrachloro-4,4--Bipyridine **10**

To a solution of diisopropylamine (27.5 mmol, 3.85 mL) in THF (200 mL) at −40 ◦C was added a solution of *n*-BuLi (1.6 M in hexanes, 27.5 mmol, 17.2 mL) and the mixture was stirred for 20 min. A solution of 3,5-dichloropyridine **11** (50 mmol, 7.4 g) in THF (100 mL) was added during 1 h while maintaining the temperature close to −40 ◦C. The temperature of the cloudy solution was slowly raised to −15 ◦C (during 1h) to give a homogeneous dark red solution. The temperature was lowered to −78 ◦C then a solution of I2 (30 mmol, 7.62 g) in THF (50 mL) was slowly added. After 10 min at −78 ◦C, the temperature was raised to room temperature and the reaction was quenched by the addition of aqueous saturated solution of Na2S2O3 (100 mL). Water was added (200 mL) and the mixture was extracted three times with ethyl acetate (3 × 200 mL). The organic phases were combined, washed with brine (100 mL) and dried over MgSO4. After concentration, the crude compound was purified by chromatography on silica gel (cyclohexane/ethyl acetate 95/5) to give 4,4--bipyridine **10** (3.64 g) and compound **12** (2.7 g). Compound **12** was diluted with ethyl acetate (200 mL) and I2 (18 mmol, 4.57g) was added and the mixture was stirred overnight at room temperature in an open flask. The reaction was quenched by the addition of aqueous saturated solution of Na2S2O3 (25 mL). Water was added (50 mL) and the organic phases washed with brine (100 mL) and dried over anhydrous MgSO4. After concentration, the crude compound was purified by chromatography on silica gel (cyclohexane/ethyl acetate 95/5) to give 4,4--bipyridine **10** (2.4 g). A total mass of 6.04 g of **10** was obtained which corresponds to an overall yield of 82%. The NMR data for **10** are in complete agreement with the literature [22].

*3,3*-*,5,5*-*-Tetrachloro-1,4-dihydro-4,4*-*-bipyridine* (**12**). Yellow solid, mp 155–157 ◦C. 1H NMR (500 MHz, CD3COCD3) δ 8.57 (s, 1H), 8.56 (s, 1H), 7.55 (s, 1H, NH), 6.56 (s, 1H), 6.55 (s, 1H), 5.87 (s, 1H). 13C NMR (126 MHz, CD3COCD3) δ 150.7, 148.3, 142.5, 134.3, 134.0, 127.7, 102.8, 50.6. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H6Cl5N2 294.9285, found: 294.9327 (Figure S1).

#### 2.2.2. 3,3-,5,5--Tetrachloro-[4,4--Bipyridine] 1-Oxide **13**

4,4--Bipyridine **10** (12.38 mmol, 3.64 g) was dissolved in CH2Cl2 (62 mL) at room temperature. *m*-CPBA (*m*-chloro-perbenzoic acid) (77% purity, 12.38 mmol, 2.8 g) was added and the mixture was stirred for 24 h. After dilution with CH2Cl2 (140 mL), the mixture was washed with NaOH 1M (2 × 100 mL) and with brine. The organic phase was dried over MgSO4, concentrated and purified by chromatography on silica gel (CH2Cl2/methanol 99/1) to give respectively the starting compound **10** (850 mg, 23%), compound **13** (2.0 g, 54% Yield) and the bis *N*-oxide **14** (925 mg, 23%).

*3,3*-*,5,5*-*-Tetrachloro-[4,4*-*-bipyridine] 1-oxide* (**13**). White solid, mp 218–220 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.68 (s, 2H), 8.31 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 148.0, 139.0, 138.3, 132.9, 132., 130.2. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H5Cl4N2O 308.9150, found: 308.9153 (Figure S2).

*3,3*-*,5,5*-*-Tetrachloro-[4,4*-*-bipyridine] 1,1*-*-dioxide* (**14**). White solid, mp 270–272 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.31 (s, 4H). 13C NMR (126 MHz, CDCl3) δ 138.3, 133.7, 128.9. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H5Cl4N2O2 324.9100, found: 324.9095 (Figure S3).

#### 2.2.3. 2-Bromo-3,3-,5,5--Tetrachloro-4,4--Bipyridine **15**

3,3-,5,5--Tetrachloro-[4,4--bipyridine] 1-oxide **13** (6.37 mmol, 1.91 g) was dissolved in dibromoethane (15 mL) and triethylamine (12.73 mmol, 1.7 mL) was added. The mixture was cooled to −40 ◦C and oxalyl bromide (12.73 mmol, 1.8 mL) was slowly added while maintaining the temperature below −35 ◦C. After stirring 1h at −40 ◦C, methanol (1 mL) was added and temperature was raised to room temperature. A saturated solution of ammonium chloride (20 mL) was added and the mixture was extracted with CH2Cl2 (3 × 50 mL). The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude compound was purified by chromatography on silica gel (pentane/CH2Cl2 7/3 to 2/1) to give pure 4,4--bipyridine **15** (2.18 g, 94% yield). White solid, mp 100–102 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.68 (s, 2H), 8.48 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 148.1, 147.2, 142.1, 140.7, 140.4, 132.9, 130.9, 130.6. HRMS (ESI-TOF) [M + H]+ *m*/*z*: Calcd. for C10H4BrCl4N2 370.8306, found: 370.8302 (Figure S4).

#### 2.2.4. 3,3-,5,5--Tetrachloro-[4,4--Bipyridine]-2-Carbonitrile **16**

3,3-,5,5--Tetrachloro-[4,4--bipyridine] 1-oxide **13** (1 mmol, 310 mg) was dissolved in acetonitrile (2 mL) and triethylamine (0.5 mL). Trimethylsilylcyanide (3 mmol, 375 μL) was slowly added and the mixture was refluxed at 100 ◦C for 24 h. The temperature lowered to 0 ◦C, a solution of NaOH (5 M, 20 mL) was slowly added and the mixture was extracted with CH2Cl2 (3 × 30 mL). The organic phase was dried over MgSO4, filtered and concentrated under vacuum. The crude compound was purified by chromatography on silica gel (pentane/ethyl acetate 7/3) to give 4,4--bipyridine **16** (267 mg, 83% yield). White solid, mp 99–101 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.71 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 149.1, 148.2, 141.9, 138.7, 135.40, 135.35, 132.3, 130.9. HRMS (ESI-TOF) [M + H]+ *m*/*z*: Calcd. for C11H3Cl4N3 317.9154, found: 317.9151 (Figure S5).

#### 2.2.5. 2-Bromo-3,3-,5,5--Tetrachloro-[4,4--Bipyridine] 1-Oxide **17**

2-Bromo-3,3-,5,5--tetrachloro-4,4--bipyridine **15** (5.36 mmol, 2.0 g) was dissolved in CH2Cl2 (28 mL) at room temperature. *m*-CPBA (77% purity, 11 mmol, 2.47 g) was added and the mixture was stirred for 60 h. After dilution with CH2Cl2 (100 mL), the mixture was washed with NaOH 1M (2 × 80 mL) and with brine. The organic phase was dried over MgSO4, concentrated and purified by chromatography on silica gel (CH2Cl2/methanol 99/1) to give compound **17** (1.72 g, 83%). White solid, mp 212–214 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.48 (s, 1H), 8.31 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 147.2, 141.1, 140.8, 138.4, 133.6, 132.6, 131.3, 130.4. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H3BrCl4N2O 386.8256, found: 386.8265 (Figure S6).

#### 2.2.6. 3,3-,5,5--Tetrachloro-2--Cyano-[4,4--Bipyridine] 1-Oxide **18**

Procedure 2.2.5. was used starting from 3,3-,5,5--tetrachloro-[4,4--bipyridine]-2-carbonitrile **16** (0.5 mmol, 160 mg) and *m*-CPBA (77% purity, 1 mmol, 224 mg) in CH2Cl2 (2.5 mL). After purification by chromatography on silica gel (CH2Cl2/methanol 98/2), compound **18** was obtained (158 mg, 94%). White solid, mp 193–195 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.32 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 149.2, 141.0, 138.5, 136.2, 136.1, 132.7, 132.3, 128.4, 113.9. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C11H3Cl4N3O 333.9103, found: 333.9091 (Figure S7).

#### 2.2.7. 2-Bromo-2-,3,3-,5,5--Pentachloro-4,4--Bipyridine **19**

Procedure 2.2.3. was used starting from 2-bromo-3,3-,5,5--tetrachloro-[4,4--bipyridine] 1- oxide **17** (0.179 mmol, 70 mg), oxalyl chloride (0.358 mmol, 31 μL), triethylamine (0.358 mmol, 50 μL) in CH2Cl2 (0.5 mL). After purification by chromatography on silica gel (pentane/ethyl acetate 97/3), compound **19** was obtained (51 mg, 70%). White solid, mp

119–121 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H), 8.49 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 148.7, 147.3, 146.9, 142.9, 142.2, 140.8, 132.6, 130.3, 129.9, 129.8. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H2BrCl5N2 404.7917, found: 404.7921 (Figure S8).

#### 2.2.8. 2-,3,3-,5,5--Pentachloro-[4,4--Bipyridine]-2-Carbonitrile **20**

Procedure 2.2.3. was used starting from 3,3-,5,5--tetrachloro-[4,4--bipyridine]-2-carbonitrile **16** (0.179 mmol, 60 mg), oxalyl chloride (0.358 mmol, 31 μL), triethylamine (0.358 mmol, 50 μL) in CH2Cl2 (0.5 mL). After purification by chromatography on silica gel (pentane/ethyl acetate 95/5), compound **20** was obtained (45 mg, 71%). Colorless syrup. 1H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.53 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 149.2, 149.0, 147.1, 142.1, 141.2, 135.1, 132.4, 129.9, 129.7, 113.9. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C11H2Cl5N3 351.8764, found: 351.8774 (Figure S9).

#### 2.2.9. 3,3-,5,5--Tetrachloro-2--Iodo-[4,4--Bipyridine] 1-Oxide **21**

In a dry tube were placed 2-bromo-3,3-,5,5--tetrachloro-[4,4--bipyridine] 1-oxide **17** (4.11 mmol, 1.6 g), NaI (8.22 mmol, 1.23 g), CuI (0.411 mmol, 78 mg) and (rac)-*trans*cyclohexane-1,2-diamine (0.822 mmol, 117 mg). The tube was evacuated and filled with argon before addition of degassed dioxane (15 mL). The tube was sealed with a screw cap and heated at 120 ◦C for 60 h. After cooling to room temperature, NH4OH (20 mL) and H2O (40 mL) were added then the product was extracted with dichloromethane (3 × 50 mL). After drying over MgSO4, filtration and concentration, the crude compound was purified by chromatography on silica gel (CH2Cl2/methanol 99/1) to give compound **21** (1.29 g, 72%). White solid, mp 244–246 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.47 (s, 1H), 8.30 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 147.8, 139.2, 138.3, 138.0, 132.6, 131.8, 130.7, 119.4. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H3Cl4IN2O 434.8117, found: 434.8109 (Figure S10).

#### 2.2.10. 2,3,3-,5,5--Pentachloro-2--Iodo-4,4--Bipyridine **22**

Procedure 2.2.3. was used starting from 3,3-,5,5--tetrachloro-2--iodo-[4,4--bipyridine] 1- oxide **21** (0.179 mmol, 78 mg), oxalyl chloride (0.358 mmol, 31 μL), triethylamine (0.358 mmol, 50 μL) in CH2Cl2 (0.5 mL). After purification by chromatography on silica gel (pentane/ethyl acetate 97/3), compound **22** was obtained (65 mg, 80%). White solid, mp 140–142 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.48 (s, 1H), 8.49 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 148.7, 147.9, 146.9, 143.1, 140.4, 137.0, 130.8, 129.8, 129.7, 119.5. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H2Cl5IN2 452,7778, found: 452.7749 (Figure S11).

#### 2.2.11. 2--Bromo-3,3-,5,5--Tetrachloro-[4,4--Bipyridine]-2-Carbonitrile **23**

Procedure 2.2.3. was used starting from 3,3-,5,5--tetrachloro-2--cyano-[4,4--bipyridine] 1-oxide **18** (0.297 mmol, 100 mg), oxalyl bromide (0.595 mmol, 85 μL), triethylamine (0.595 mmol, 80 μL) in CH2Br2 (1 mL). After purification by chromatography on silica gel (pentane/ethyl acetate 95/5), compound **23** was obtained (107 mg, 90%). White solid, mp 131–133 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.52 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 149.2, 147.4, 142.2, 141.0, 140.7, 135.06, 135.05, 132.5, 132.4, 130.2, 113.9. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C11H2BrCl4N3 395.8259, found: 395.8277 (Figure S12).

#### 2.2.12. 2-Bromo-3,3-,5,5--Tetrachloro-2--Iodo-4,4--Bipyridine **24**

Procedure 2.2.3. was used starting from 3,3-,5,5--tetrachloro-2--iodo-[4,4--bipyridine] 1-oxide **21** (1.74 mmol, 760 mg), oxalyl bromide (3.49 mmol, 496 μL), triethylamine (3.49 mmol, 463 μL) in CH2Br2 (4.2 mL). After purification by chromatography on silica gel (pentane/CH2Cl2 7/3), compound **24** was obtained (710 mg, 81%). White solid, mp 165–167 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.414 (s, 1H), 8.410 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 147.9, 147.3, 142.6, 140.8, 140.5, 137.0, 132.5, 130.8, 130.2, 119.5. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C10H2BrCl4IN2 496.7273, found: 496.7242 (Figure S13).

#### 2.2.13. 3,3-,5,5--Tetrachloro-2--Iodo-[4,4--Bipyridine]-2-Carbonitrile **25**

Procedure 2.2.4. was used starting from 3,3-,5,5--tetrachloro-2--iodo-[4,4--bipyridine] 1- oxide **21** (0.395 mmol, 172 mg), acetonitrile (1.5 mL), triethylamine (0.4 mL) and trimethylsilylcyanide (1.18 mmol, 150 μL). After purification by chromatography on silica gel (pentane/ethyl acetate 95/5), compound **25** was obtained (110 mg, 62%). White solid, mp 144–146 ◦C. 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.51 (s, 1H). 13C NMR (126 MHz, CDCl3) δ 149.2, 148.0, 142.4, 138.9, 137.0, 135.1, 135.0, 132.4, 130.7, 119.7, 114.0. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C11H2Cl4IN3 443.8120, found: 443.8121 (Figure S14).

#### 2.2.14. 2-Bromo-3,3-,5,5--Tetrachloro-2--Phenyl-4,4--Bipyridine **26**

In a dry tube were placed 2-bromo-3,3-,5,5--tetrachloro-2--iodo-4,4--bipyridine **24** (0.3 mmol, 151.5 mg), Pd(PPh3)4 (0.015 mmol, 17.3 mg), phenylboronic acid (0.3 mmol, 36.6 mg) and K3PO4 (0.3 mmol, 64 mg). The tube was evacuated and filled with argon before addition of degassed mixture of toluene/water/ethanol (6:1:1, 4 mL). The tube was sealed with a screw cap and heated at 100 ◦C for 15 h. The cooled reaction mixture was diluted with water (10 mL) and extracted with CH2Cl2 (3 × 15 mL). After drying over MgSO4, filtration and concentration, the crude compound was purified by chromatography on silica gel (pentane/ethyl acetate 98/2) to give compound **26** (122 mg, 89%). Colorless syrup. 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.49 (s, 1H), 7.78–7.25 (m, 2H), 7.52–7.46 (m, 3H). 13C NMR (126 MHz, CDCl3) δ 156.2, 147.5, 147.2, 143.1, 141.7, 140.7, 137.0, 132.8, 130.6, 129.6, 129.5, 129.1, 128.7, 128.4. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C16H7BrCl4N2 446.8619, found: 446.8625 (Figure S15).

## 2.2.15. 2-Bromo-2--(4-((Tert-Butyldimethylsilyl)oxy)phenyl)-3,3-,5,5--Tetrachloro-4,4--Bipyridine **27**

Procedure 2.2.14. was used starting from 2-bromo-3,3-,5,5--tetrachloro-2--iodo-4,4-- bipyridine **24** (0.3 mmol, 151.5 mg), Pd(PPh3)4 (0.015 mmol, 17.3 mg), 4-((tert-butyldimethylsilyl)oxy)phenyl boronic acid (0.3 mmol, 75.7 mg) and K3PO4 (0.3 mmol, 64 mg). After purification by chromatography on silica gel (pentane/ethyl acetate 98/2), compound **27** was obtained (137 mg, 79%). Colorless syrup. 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H), 8.49 (s, 1H), 7.69 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 1.00 (s, 9H), 0.24 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 157.1, 155.8, 147.3, 147.2, 143.3, 141.6, 140.7, 132.9, 131.1, 130.7, 130.0, 128.5, 128.4, 120.0, 25.8, 18.4, −4.2. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C22H22BrCl4N2OSi 576.9433, found: 576.9428 (Figure S16).

2.2.16. 2---(4-((Tert-Butyldimethylsilyl)oxy)phenyl)-3-,3--,5-,5---Tetrachloro-4,2-:4-,4--- Terpyridine **31**

Procedure 2.2.14. was used starting from 2-bromo-3,3-,5,5--tetrachloro-2--iodo-4,4-- bipyridine **24** (0.143 mmol, 82.5 mg), Pd(PPh3)4 (0.007 mmol, 8.3 mg), 4-pyridine boronic acid (0.215 mmol, 31 mg) and K3PO4 (0.215 mmol, 46 mg). After purification by chromatography on silica gel (pentane/ethyl acetate 7/3), compound **31** was obtained (60 mg, 73%). Colorless syrup. 1H NMR (500 MHz, CDCl3) δ 8.80 (s, 1H), 8.78 (d, J = 5.5 Hz, 2H), 8.74 (s, 1H), 8.76–8.66 (m, 4H), 6.95 (d, J = 8.5 Hz, 2H), 1.00 (s, 9H), 0.24 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 157.1, 155.9, 153.2, 150.1, 147.9, 147.3, 144.7, 142.9, 141.8, 131.09, 131.05, 130.1, 129.5, 128.7, 128.6, 124.0, 120.0, 25.8, 18.4, −4.2. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C27H26Cl4N3OSi 576.0594, found: 576.0589 (Figure S17).

#### 2.2.17. 4-(3-,3--,5-,5---Tetrachloro-[4,2-:4-,4---Terpyridin]-2---yl)phenol **32**

2---(4-((Tert-butyldimethylsilyl)oxy)phenyl)-3-,3--,5-,5---tetrachloro-4,2-:4-,4---terpyridine **31** (0.078 mmol, 45 mg) was dissolved in THF (2 mL) and the solution was cooled to 0 ◦C. A tetra-butylammonium fluoride (TBAF) solution (1M in THF, 0.117 mmol, 117 μL) was slowly added and the mixture was stirred at 0 ◦C for 30 min. After addition of a saturated solution of NH4Cl (1 mL), the temperature was raised to ambient. Water was added (5 mL) and the mixture was extracted with CH2Cl2 (3 × 10 mL). The organic phase was washed

with brine, dried over MgSO4, filtered and concentrated. After filtration on silica gel (ethyl acetate), compound **32** was obtained (36 mg, 100%). White solid, mp 254–256 ◦C. 1H NMR (500 MHz, DMSO-*d6*) δ 9.998 (broad s, 1H), 9.11 (s, 1H), 8.99 (s, 1H), 8.77 (d, J = 6.0 Hz, 2H), 7.73 (d, J = 6.0 Hz, 2H), 7.62 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H). 13C NMR (126 MHz, DMSO-*d6*) δ 158.8, 155.4, 153.1, 150.0, 148.2, 147.5, 143.9, 142.0, 141.1, 131.1, 130.2, 128.4, 127.6, 127.4, 127.2, 115.1. HRMS (ESI-TOF) [M + H]+ *m/z*: Calcd. for C21 H12Cl4N3O 461.9729, found: 461.9701 (Figure S18).

#### *2.3. Single Crystal X-ray Diffraction*

#### 2.3.1. Crystallizations and Analysis

Suitable crystals of compounds **13**, **18** and **24** were obtained by slow evaporation of a dichloromethane/hexane (1:1) solution. The solid-state structures were determined by single crystal X-ray diffraction at low temperature (T = 100 K).

#### 2.3.2. Crystal Data

CCDC 2077648, 2077649 and 2077650 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac. uk/conts/retrieving.html (accessed on 14 April 2021) (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).

Crystal Data for **24** C10 H2BrCl4IN2 (M = 498.75 g/mol): Monoclinic, space group *P*21/*c* (no. 14), a = 7.9297(2) Å, b = 15.6388(4) Å, c = 11.4938(3) Å, β =105.632(2)◦, V = 1372.64(6) Å3, Z = 4, T = 100(2) K, μ(MoK α) = 6.002 mm−1, Dcalc = 2.413 g/cm3, 51767 reflections measured (2.254◦ ≤ Θ ≤ 36.150◦), 6552 unique (Rint = 0.0298), which were used in all calculations. The final R1 was 0.0239 (I > 2σ(I)) and wR2 was 0.0548 (all data). CCDC 2077649.

Crystal Data for **18** C11 H3Cl4N3 (M = 334.96 g/mol): Monoclinic, space group *P*21/*c* (no. 14), a = 7.2662(2) Å, b = 12.0325(4) Å, c = 14.4319(5) Å, β = 93.491(3)◦, V = 1259.45(7) Å3, Z = 4, T = 100(2) K, μ(MoK α) = 0.931 mm−1, Dcalc = 1.767 g/cm3, 48102 reflections measured (2.205◦ ≤ Θ ≤ 33.667◦), 5000 unique (Rint = 0.0424), which were used in all calculations. The final R1 was 0.0323 (I > 2σ(I)) and wR2 was 0.0732 (all data). CCDC 2077650.

Crystal Data for **13** C10 H4Cl4N2 (M = 309.95 g/mol): Monoclinic, space group *P*21/*c* (no. 14), a = 7.2697(2) Å, b = 11.5462(3) Å, c = 14.0808(4) Å, β = 97.804(3)◦, V = 1170.96(6) Å3, Z = 4, T = 100(2) K, μ(MoK α) = 0.991 mm−1, Dcalc = 1.758 g/cm3, 48887 reflections measured (2.290◦ ≤ Θ ≤ 37.162◦), 6021 unique (Rint = 0.0444), which were used in all calculations. The final R1 was 0.0384 (I > 2σ(I)) and wR2 was 0.0840 (all data). CCDC 2077648.

#### *2.4. Isolated Molecule Calculations*

Molecular structures of **24**, **18** and **13** were optimized with Gaussian09 software at DFT level of theory using the B3LYP functional completed with D3 dispersion correction and the Def2TZVPP basis set. Frequency calculations were performed in order to check that true energy minimum were obtained. Electrostatic maps (Figure 2, Figures S31 and S32) were drawn using the AIMAll software, and locations of ESP extrema VS,max were searched using MultiWFN program (Table S4). Integrated atomic charges (AIMAll) are depicted on Figures S33–S35.

**Figure 2.** Electrostatic potential mapped on the *ρ* = 0.002 a.u. electron density isosurface of **24**. Coloring from red = −0.05 a.u. to blue = +0.05 a.u. Black arrows point to some representative electropositive σ- and π-holes.

## **3. Results**

#### *3.1. Improved Synthesis of 3,3*-*,5,5*-*-Tetrachloro-4,4*-*-Bipyridine* **10**

The starting 4,4--bipyridine **10** was already prepared by us with 44% yield by dimerization of 3,5-dichlopyridine **11** [22]. When the synthesis was reproduced on large scale (50 mmol), the yield was improved to 50%. Moreover, during the chromatographic purification, the more polar non-rearomatized compound **12** was also isolated with 40% yield. Compound **12** could be oxidized with I2 in ethyl acetate at room temperature for 12 h, furnishing **10** with 90% yield. Overall, the yield of **10** in the dimerization process could be increased to 82% yield (Scheme 1).

**Scheme 1.** Improved synthesis of 3,3-,5,5--tetrachloro-4,4--bipyridine **10**.

#### *3.2. Desymmetrization of 3,3*-*,5,5*-*-Tetrachloro-4,4*-*-Bipyridine* **10**

The mono *N*-oxidation of 4,4--bipyridine **10** was performed using 1 equivalent of *m*-CPBA in CH2Cl2 (0.2 M) at room temperature. After treatment using NaOH 1M to remove the benzoic acid, the crude mixture was purified by chromatography on silica gel to give the expected mono-oxidized 4,4--bipyridine **13** with 54% yield. Unreacted 4,4-- bipyridine **10** (23%) as well as doubly oxidized 4,4--bipyridine **14** (23%) were also obtained after the purification. Based on the recovery of the starting 4,4--bipyridine **10**, the yield of **14** was of 75%. The *N*-oxide **13** could be further transformed to the pentahalogenated 4,4--bipyridine **15** in very good yields by using oxalyl bromide with Et3N in CH2Br2 at low temperature [23], and to the cyano derivative **16** by using TMSCN/Et3N in refluxing dioxane [24] (Scheme 2).

**Scheme 2.** Mono-oxidation of 4,4--bipyridine **10** and its functionalization.

#### *3.3. New Chiral Non-Symmetrical 4,4*-*-Bipyridines Based on the 3,3*-*,5,5*-*-Tetrachloro-4,4*-*- Bipyridine Core*

4,4--Bipyridines **15** and **16** were submitted to a new sequence of *N*-oxidation/halogenation in order to access to novel chiral non-symmetrical 4,4--bipyridines. Thus, regioselective *N*-oxidation of the less substituted ring of **15** and **16** furnished, respectively, 4,4--bipyridine *N*-oxides **17** and **18** in high yields. Chlorination of *N*-oxides **17** and **18** using oxalyl chloride with Et3N in CH2Cl2 at low temperature [23] afforded 4,4--bipyridines **19** and **20** in good yields. Alternatively, *N*-oxide **17** was transformed by a copper-catalyzed Finkelstein reaction [25] to the iodinated 4,4--bipyridine *N*-oxide **21** which was further submitted to the chlorination to provide 4,4--bipyridine **22**. *N*-oxides **18** and **21** were also brominated as described in Scheme 2 to give two other chiral non-symmetrical 4,4--bipyridines **23** and **24** in good yields (Scheme 3). Finally, 4,4--bipyridine **21** was transformed in good yield to the iodinated 4,4--bipyridine **25** by using TMSCN/Et3N in refluxing dioxane.

**Scheme 3.** Synthesis of six new chiral non-symmetrical 4,4--bipyridines.

#### *3.4. Cross-Coupling Reactions with 4,4*-*-Bipyridine* **24**

4,4--Bipyridine **24** bearing two different halogens (Br and I) in 2 and 2--positions was chosen to perform selective cross-coupling reactions. In particular, 4,4--bipyridines **28** and **30,** which were recently described as good transthyretin fibrillogenesis inhibitors [17], could be obtained in excellent yields and with very high purity by successive Suzuki and Finkelstein reactions, followed by silyl group deprotection in the case of **30**. Moreover, their enantiomers could be separated by HPLC on chiral stationary phase [26,27]. High selectivity for mono-coupling products **26** and **27** was obtained by using the Suzuki coupling [28] with one equivalent of arylboronic acid, letting the C–Br bond untouched for a further coupling reaction such as the copper-catalyzed Finkelstein Br/I exchange [18]. Moreover, a new Suzuki cross-coupling of 4,4--bipyridine **27** with 4-pyridylboronic acid delivered compound **31,** which, after silyl group deprotection, furnished the chiral 4,4--bipyridine **32,** bearing two different functional groups (Scheme 4).

**Scheme 4.** Cross-coupling reactions with 4,4--bipyridine **24**.

#### *3.5. X-ray Diffraction Analysis*

One non-symmetrical 4,4--bipyridine (**24**) and two 4,4--bipyridine *N*-oxydes (**13** and **18**) solid state structures were determined by single X-ray diffraction.

2-Bromo-3,3-,5,5--tetrachloro-2--iodo-4,4--bipyridine **24** crystallizes in *P*21/*c* space group with one molecule in the asymmetric unit. A positional disorder (corresponding to two different orientations of the molecule) exchanges the Br and I halogen atom positions, the major component having a population of 0.7441(13). According to the energy packing analysis performed with CrystalExplorer software [29], the solid-state structure of **24** is primarily determined by the formation of intermolecular interactions with a predominance of stabilizing dispersion over electrostatic contributions (Table S1, entries 1–6). These contributions involve first the formation of strongly bound dimers centered on an inversion center where π··· π stacking occurs (N7-pyridine interplane distance of 3.272 Å) (Table S1, entry 1 and Figure S19), with a complementary electrostatic interaction between the electropositive C12-H12 group (integrated atomic charges of Q(C12) = +0.59 and Q(H12) = +0.06) and the electronegative crown of Br1 (Q = −0.02), as evidenced from the electrostatic potential mapped on the isodensity molecular surface (Figure 2). In this dimer, the electronegative region about N7 is also near the electropositive area of Cl1, although the σ-hole of the halogen is not optimally oriented (C3-Cl1··· N7 = 127.59◦). The geometry of the next two

intermolecular interactions (x, −y + 3/2, z − 1/2 and 1 + x, −y + 3/2, z + 1/2, Table S1, entries 2–3) can also be understood from electrostatic point of view, since electronegative regions (respectively Cl3 with Q(Cl3) = −0.17 and N1 with Q(N1) = −1.12) are close to electropositive area (respectively C6 with Q(C6) = +0.59 and C12 with Q(C12) = +0.59) (Figures S20 and S21).

The most remarkable feature of the molecular packing is the formation of a fourmembered ring involving four I/Br atoms engaged in type-II halogen··· halogen bonds (Figure 3). Although the I/Br positional disorder can induce bias in the modeled interatomic distances, these latter show a clear interpenetration of the van der Waals spheres and an interaction geometry typical of type-II hal··· hal bonds [30,31] (I1··· Br1 = 3.564(2) Å RR = 0.93; C2-I1··· Br1 = 170.22(7)◦; I1··· Br1-C8 = 114.6(1)◦; Br1···I1 = 3.716(3) Å RR = 0.97; C8- Br1··· I1 = 170.4(1)◦; Br1··· I1-C2 = 81.45(6)◦)). Noticeably, the shortest type-II hal··· hal bond is obtained when the σ-hole of the I atom points toward the crown of the Br atom; indeed, the σ-hole is more electropositive for iodine than for bromine (Figure 2 and Table S4), whereas the electronegative crown is more pronounced for the latter. From the intermolecular energy decomposition (Table S1), these interactions appear with predominant electrostatic contribution (entry 7: −1 + x, y, −1 + z), but also with major dispersion contribution along with significant electrostatic contribution (entry 4: 1 − x, 1 − y, 1 − z). This is due to the fact that in the former dimer the molecules are well separated (interacting only through the halogen atoms), whereas in the latter a close proximity of polarizable halogen atoms and aromatic rings is observed.

**Figure 3.** Four-membered ring involving four I/Br atoms (x, y, z; −1 + x, y, −1 + z; −x, 1 − y, −z; 1 − x, 1 − y, 1 − z) engaged in type-II halogen··· halogen bonds in the crystal structure of **24**. Representative distances are given in Å. Only the major component disorder is shown.

In order to rationalize the observed positional disorder that involves the iodine and bromine atoms, DFT calculations were undertaken on the isolated tetramer centered about the type-II hal··· hal bond's four-membered ring (Figure S36). From the results (Table S5), it appears that the most stable configuration is obtained for the observed largest component disorder (labelled IBrIBr), where the halogen atoms alternate in the sequence Hal1=I, Hal2=Br, Hal3=I, Hal4=Br, as depicted on Figure 3. However, the other configurations are not excessively less stabilizing, with the most unfavorable case (IIII) being only 8.92 kJ/mol higher in energy. Boltzmann populations calculated, taking into account all the possible configurations, finally lead to an equivalent average disorder population of 0.675, in remarkable qualitative agreement with the refined population parameter (0.7441(13)). This may indicate that the observed dissymmetry in the disorder population (i.e., different from 50/50) results from the preferential interaction of the σ-hole of iodine atom toward the crown of bromine at the short distance, at the expense of the reverse situation (interaction of bromine σ-hole toward the crown of iodine).

*N*-oxides **13** and **18** are isostructural, crystallizing in *P*21/*c* space group with similar unit cell parameters. Indeed, they present similar crystal packing, with notable differences only about the substituent in 2 position (-H in **13**, -CN in **18**) (Figures S22 and S23). The analysis of intermolecular interaction energies (Tables S2 and S3) shows that in both structures the main interaction (entry 1) corresponds to the formation of a strong cyclic R2,2(8) hydrogen bond motif about an inversion center, involving the oxygen atom of the *N*-O as the acceptor (Figure 4; **18**: C12-H12··· O13 = 2.17 Å; 160◦; **13**: 2.07 Å; 162◦).

**Figure 4.** Packing in the solid-state structure of **18**. Interaction between x, y, z, and −1 + x, 1−y, −z molecules. Representative distance is given in Å and angle in ◦.

The main differences between the two structures concern the next two most intense interactions involving 2 − x, 1 − y,1 − z and 1 − x, 1 − y,1 − z molecules. The first dimer is strongly stabilizing in **18**, with the cyano groups of the two neighboring molecules arranged in a head-to-tail manner (Figure S24), associated with a large electrostatic component (Table S2, entry 2); on the contrary, in **13** the corresponding molecules does not present any contact below van der Waals limit and dispersion is the main stabilizing contribution to the interaction (Table S3, entry 2 and Figure S25). The second dimer (with 1 − x, 1 − y,1 − z, Tables S2 and S3, entry 3) is twice more stabilizing in **18** than in **13** and involves a contact at van der Waals limit between the Cl1 and C14 atoms of the cyano group, the crown of the halogen atom pointing in the direction of the positively charged carbon atom (C3- Cl1··· C14 = 3.442 Å, 110.04◦; Q(C14) = 0.94) (Figure S26); in such a way, both electrostatic and dispersion components are more stabilizing in **18**, while in **13** a C3-Cl1··· Cl1 contact at van der Waals limit (3.490 Å, 78.83◦) implies the crown of both halogen atoms in a unfavorable disposition (Figure S27).

The next interaction (Tables S2 and S3, entry 5; x, −y + 3/2, z − 1/2) evidences also some differences between the structures, although to a lesser extent. In both structures this dimer displays a halogen bond between the σ-hole of Cl3 and the O13 oxygen atom of the *N*-oxide group (**18**: C9-Cl3··· O13 = 3.152 Å RR = 0.96, 156.54◦; **13**: 3.021 Å, RR = 0.92, 157.94◦; Figures S28 and S29). However, in **18** a second halogen bond participates in the stabilization, the σ-hole of Cl2 being properly oriented toward the nitrogen atom of the cyano group (C5-Cl2··· N15 = 3.128 Å, RR = 0.95, 156.31◦).

The last significant interaction implies 1 − x, y + 1/2, −z + 1/2 molecule (Tables S2 and S3, entry 6) and is slightly more stabilizing for **13** than for **18** due to a more negative electrostatic contribution. In this latter Cl2 atom presents three contacts at distances smaller than van der Waals limits with this neighbor, namely with N7 (3.179 Å, RR = 0.96), C8 (3.380 Å, RR = 0.98) and C12 (3.426 Å, RR = 0.99), but with an orientation that does not involves the halogen σ-hole (C5-Cl2··· N7 = 109.10◦) (Figure S30). The electrostatic stabilization may then result from the head-to-tail relative orientation of the two molecules which bear a dipole moment parallel to their long N-N axis in the case of **13** (1.87 D), whereas in **18** the molecular dipole moment (3.42 D) is almost parallel to the –CN group. In this situation, the neighboring molecules have almost orthogonal dipole moments and thus a reduced overall electrostatic stabilization.

## **4. Discussion**

The synthesis of 3,3-,5,5--tetrachloro-4,4--bipyridine **10** [22] was greatly improved by adjusting the amount of LDA to 0.55 equivalent with regard to 3,5-dichloropyridine **11**. Moreover, a large quantity (44%) of dihydropyridine **12** was also isolated during the reaction, confirming the proposed mechanism for the dimerization process [22]. Indeed, after deprotonation of half equivalent of **11**, the nucleophilic lithiated **11-Li** added in 4- position of the remaining half equivalent of **11** to give **12-Li,** which after oxidation with iodine and hydrolysis delivered **10** and non-oxidized **12** (Figure 5A). After purification, dihydropyridine **12** was very stable in the solid state with no noticeable oxidation after three months on the bench at ambient temperature. In acetone solution, a slow oxidation occurred with almost complete formation of 4,4--bipyridine **10** after 10 days, as shown by 1H NMR (Figure 5B).

**Figure 5.** (**A**) Proposed mechanism for the formation of 4,4--bipyrine **10;** (**B**) 1H NMR spectra showing oxidation of dihydropyridine **12** in acetone over time.

The desymmetrization route of 4,4--bipyridines used in this study is based on the *N*-oxidation of one of the two pyridine nitrogens in the presence of one equivalent of m-CPBA. *N*-oxidation of electron-deficient pyridines generally requires harsher conditions that necessitate prior *N*-activation with trifluoroacetic acid anhydride [32,33]. As expected, this first *N*-oxidation is not selective; however, the chemical nature of the three compounds of the reaction mixture (starting material with free nitrogens, mono *N*-oxide and bis *N*oxide) allowed for simple purification with easy recovery of unreacted starting material. The *N*-oxidation of the electron-deficient pentasubstituted 4,4--bipyridines **15** and **16** was highly selective with no formation of the bis *N*-oxide. The high selectivity of the reaction is due to both electronic and steric factors. Indeed, the trisubstituted pyridine ring is electronically impoverished by the three electron-withdrawing groups (Cl, Cl and Br or CN)andthe *N*-pyridineisstericallyhinderedbythesubstituentin2-position(BrorCN).

The choice of the pyridine *N*-oxides in our synthesis was also guided by the numerous known methodologies for their functionalization [21]. Halogenation and cyanation of 4,4--bipyridine *N*-oxides used in this work are examples of such reactions which efficiently provided 4,4--bipyridines functionalized in 2-position with Cl, Br and CN. A general mechanism can be proposed for these transformations (Figure 6). After activation of the *N*oxide by (COCl)2, (COBr)2 or TMSCN, the intermediate may evolve following two different paths. In path a, the generated nucleophile during the first step (X− = Cl−, Br− or CN−) attacks the carbon in 2-position followed by base-assisted elimination of RO− to give the functionalized pyridine. In path b, the deprotonation of the acidic hydrogen in 2-position first occurs to generate a carbene intermediate which is trapped by the nucleophile with concomitant elimination of RO−.

**Figure 6.** Proposed mechanism for the functionalization of pyridine *N*-oxides.

The installed functions in 2,2--positions represent a potential entry to new functional groups by nucleophilic substitution of the chlorine, hydrolysis of the cyano group and crosscoupling reaction with the C-Br bond. In the latter case, we have used the copper-catalyzed Finkelstein reaction to exchange bromine by iodine, and the Suzuki reaction to introduce aryl groups in 2-positions. In this regard, 4,4--bipyridine **24** possessing both bromine and iodine in 2,2--positions is a compound of choice in order to selectively introduce two different aryl groups on the 3,3-,5,5--tetrachloro-4,4--bipyridine scaffold. This was highlighted by the synthesis of 4,4--bipyridine **32** whose supramolecular arrangement in solution and in the solid state is currently under investigation in our laboratory.

## **5. Conclusions**

A new methodology for the synthesis of chiral non-symmetrical 4,4--bipyridines was developed. It is based on the successive functionalization of 2- and 2--positions of 4,4--bipyridines by using the sequence *N*-oxidation then halogenation or cyanation. Starting from 3,3-,5,5--tetrachloro-4,4--bipyridine, all the six possible non-symmetrical isomers bearing Cl, Br, I or CN in 2- and 2--positions were efficiently prepared. The isomer bearing bromine and iodine was used as the key component for the improved synthesis of biologically active 2-iodinated 4,4--bipyridines and for the preparation of 4,4--bipyridines with two different aryl groups in 2- and 2--positions. This work opens new directions toward the synthesis of non-symmetrical ligands for the design of supramolecular materials, such as coordination polymers and metal-organic frameworks (MOFs).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/compounds1020006/s1, Figure S1: 3,3-,5,5--Tetrachloro-1,4-dihydro-4,4--bipyridine (**12**), Figure S2: 3,3-,5,5--Tetrachloro-[4,4--bipyridine] 1-oxide (**13**), Figure S3: 3,3-,5,5--Tetrachloro-[4,4-- bipyridine] 1,1--dioxide (**14**), Figure S4: 2-Bromo-3,3-,5,5--tetrachloro-4,4--bipyridine (**15**), Figure S5: 3,3-,5,5--Tetrachloro-[4,4--bipyridine]-2-carbonitrile (**16**), Figure S6: 2-Bromo-3,3-,5,5--tetrachloro- [4,4--bipyridine] 1-oxide (**17**), Figure S7: 3,3-,5,5--Tetrachloro-2--cyano-[4,4--bipyridine] 1-oxide (**18**), Figure S8: 2-Bromo-2-,3,3-,5,5--pentachloro-4,4--bipyridine (**19**), Figure S9: 2-,3,3-,5,5--Pentachloro- [4,4--bipyridine]-2-carbonitrile (**20**), Figure S10: 3,3-,5,5--Tetrachloro-2--iodo-[4,4--bipyridine] 1-oxide (**21**), Figure S11: 2,3,3-,5,5--Pentachloro-2--iodo-4,4--bipyridine (**22**), Figure S12: 2--Bromo-3,3-,5,5-- tetrachloro-[4,4--bipyridine]-2-carbonitrile (**23**), Figure S13: 2-Bromo-3,3-,5,5--tetrachloro-2--iodo-4,4--bipyridine (**24**), Figure S14: 3,3-,5,5--Tetrachloro-2--iodo-[4,4--bipyridine]-2-carbonitrile (**25**), Figure S15: 2-Bromo-3,3-,5,5--tetrachloro-2--phenyl-4,4--bipyridine (**26**), Figure S16: 2-Bromo-2-- (4-((tert-butyldimethylsilyl)oxy)phenyl)-3,3-,5,5--tetrachloro-4,4--bipyridine (**27**), Figure S17: 2---(4- ((Tert-butyldimethylsilyl)oxy)phenyl)-3-,3--,5-,5---tetrachloro-4,2-:4-,4---terpyridine (**31**), Figure S18: 4-(3-,3--,5-,5---Tetrachloro-[4,2-:4-,4---terpyridin]-2---yl)phenol (32), Figure S19: Packing in the solidstate structure of **24**. Interaction between x, y, z, and 2 − x, 1 − y, 1 − z molecules. Representative distance is given in Å. Only the major component disorder is shown, Figure S20: Packing in the solidstate structure of **24**. Interaction between x, y, z, and x, −y + 3/2, z − 1/2 molecules. Representative distance is given in Å. Only the major component disorder is shown, Figure S21: Packing in the solid-state structure of **24**. Interaction between x, y, z, and 1 + x, −y + 3/2, z + 1/2 molecules. Representative distance is given in Å. Only the major component disorder is shown, Figure S22: Superimposition of the molecular environment about a central molecule (displayed as ball and sticks) showing the isostructural relationship between **18** (colored as atom type) and **13** (light gray), Figure S23: Focus on the region centered on –CN group in **18**, showing the largest differences between **18** (colored as atom type) and **13** (light gray) crystal structures, Figure S24: Packing in the solid-state structure of **18**. Interaction between x, y, z, and 2 − x, 1 − y, 1 − z molecules. Representative distances are given in Å, Figure S25: Packing in the solid-state structure of **13**. Interaction between x, y, z, and 2 − x, 1 − y, 1 − z molecules. Representative distance is given in Å, Figure S26: Packing in the solidstate structure of **18**. Interaction between x, y, z, and 1 − x, 1 − y, 1 − z molecules. Representative distances are given in Å, Figure S27: Packing in the solid-state structure of **13**. Interaction between x, y, z, and 1 + x, −y + 3/2, z + 1/2 molecules. Representative distances are given in Å, Figure S28: Packing in the solid-state structure of **18**. Interaction between x, y, z, and x, −y + 3/2, z − 1/2 molecules. Representative distances are given in Å, Figure S29: Packing in the solid-state structure of **13**. Interaction between x, y, z, and x, −y + 3/2, z − 1/2 molecules. Representative distances are given in Å, Figure S30: Packing in the solid-state structure of **13**. Interaction between x, y, z, and 1 − x, y + 1/2, −z + 1/2 molecules. Representative distances are given in Å, Figure S31: Electrostatic potential mapped on the *ρ* = 0.002 a.u. electron density isosurface of **13**. Coloring from red = −0.05 a.u. to blue = +0.05 a.u., Figure S32: Electrostatic potential mapped on the *ρ* = 0.002 a.u. electron density isosurface of **18**. Coloring from red = −0.05 a.u. to blue = +0.05 a.u., Figure S33: Integrated Bader atomic charges for isolated 24 molecule, Figure S34: Integrated Bader atomic charges for isolated **13** molecule, Figure S35: Integrated Bader atomic charges for isolated **18** molecule, Figure S36: Tertamer extracted from the experimental structure of **24** and used for the modeling of I/Br substation disorder. HalX/HalX- are I/Br or Br/I, Table S1: Intermolecular interaction energies (kJ/mol) in the packing of solid state structure of **24**. R is the distance between molecular centroids (mean atomic position) in Å, Table S2: Intermolecular interaction energies (kJ/mol) in the packing of solid state structure of **18**. Largest differences between **18** and **13** are highlighted in bold. R is the distance between molecular centroids (mean atomic position) in Å, Table S3. Intermolecular interaction energies (kJ/mol) in the packing of solid state structure of **13** Largest differences between **18** and **13** are highlighted in bold. R is the distance between molecular centroids (mean atomic position) in Å, Table S4. Electrostatic potential maxima (VS,max; kcal/mol) on the molecular surface *ρ* = 0.002 a.u. of **24**, Table S5: Type-II hal··· hal bonds four membered ring tetramer relative energies. Hal1-2-3-4 numbering corresponds to Figure S18.

**Author Contributions:** Conceptualization, V.M. and P.P.; methodology, V.M.; X-ray analysis, E.A. and E.W.; data curation, V.M.; writing—original draft preparation, V.M.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** All the data are given in the article and in the associated supporting information.

**Acknowledgments:** We thank University of Strasbourg and CNRS for financial support. "The Plateforme de Mesures de Diffraction et Diffusion des rayons X of Université de Lorraine" is thanked for providing access to crystallographic facilities. The EXPLOR mesocentre is thanked for computing facilities (Project 2019CPMXX0984).

**Conflicts of Interest:** The authors declare no conflict of interest.


**Author details**

Barbara Mazur Lublin University of Technology, Lublin, Poland

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