3.12.19. Methyl 3-Deoxy-3-*C*-(4-Phenylbenzamidomethyl)-β-d-Galactopyranoside **7l**

Compound **7l** (TLC, DCM/MeOH, 10:1, R<sup>f</sup> 0.54) was prepared according to the general procedure *3.12* from the amide **22l** (39 mg, 0.06 mmol). Obtained as a colorless oil in 67% yield (15.4 mg, 0.04 mmol) from flash column chromatography (DCM:MeOH 20:1–12:1). [α] 25 <sup>D</sup> <sup>−</sup>21.4 (c 0.7, CH3OH). <sup>1</sup>H NMR (CD3OD, 400 MHz): 7.70 (dd, 2H, *J* 6.8 Hz, *J* 2.0 Hz, Ar*H*), 7.71 (dd, 2H, *J* 6.8 Hz, *J* 2.0 Hz, Ar*H*), 7.65 (m, 2H, Ar*H*), 7.48–7.44 (m, 2H, Ar*H*), 7.40–7.35 (m, 1H, Ar*H*), 4.49 (d, 1H, *J*1,2 7.2 Hz, H-1), 3.96 (td, 1H, *J* 6.0 Hz, *J* 2.4 Hz, H-5), 3.82–3.75 (m, 2H, H-6a, H-6b), 3.73–3.62 (m, 2H, C*H2*NH), 3.54 (s, 3H, OC*H*3), 2.44–2.38 (m, 1H, H-3). <sup>13</sup>C NMR (CD3OD, 100 MHz): 170.1, 145.7, 141.2, 134.2, 130.0, 129.1, 128.8, 128.11, 128.07, 103.7, 76.0, 69.0, 68.7, 63.2, 56.9, 46.4, 37.8. HRMS calcd for C21H25NO6+H<sup>+</sup> (M+H)+: 388.1760, found: 388.1761.

3.12.20. Methyl 3-Deoxy-3-*C*-(Diphenylphosphonamidomethyl)-β-d-Galactopyranoside **8**

Compound **8** (TLC, DCM/MeOH, 10:1, R<sup>f</sup> 0.45) was prepared according to the general procedure 3.12 from the amide **23** (43 mg, 0.06 mmol). Obtained as a colorless oil in 50% yield (13.3 mg, 0.03 mmol) from flash column chromatography (DCM:MeOH 15:1–9:1). [α] 25 <sup>D</sup> <sup>−</sup>18.6 (c 0.7, CH3OH). <sup>1</sup>H NMR (CD3OD, 400 MHz): 7.40 (t, 4H, *J* 8.0 Hz, Ar*H*), 7.29–7.21 (m, 6H, Ar*H*), 4.36 (d, 1H, *J* 7.2 Hz, H-1), 3.90

(dd, 1H, *J* 4.0 Hz, *J* 2.0 Hz, H-4), 3.83 (dd, 1H, *J* 7.2 Hz, *J* 5.6 Hz, H-2), 3.79–3.76 (m, 1H, H-5), 3.71 (dd, 1H, *J*5,6a 10.4 Hz, *J*6a,6b 4.4 Hz, H-6a), 3.71 (dd, 1H, *J*5,6b 10.8 Hz, *J*6a,6b 4.4 Hz, H-6b), 3.51 (s, 3H, OC*H*3), 3.50–3.44 (m, 1H, C*H*2NH), 3.17–3.08 (m, 1H, C*H*2NH), 2.28–2.22 (m, 1H, H-3). <sup>13</sup>C NMR (CD3OD, 100 MHz): 152.2 (dd, *J* 6.2 Hz, *J* 2.7 Hz), 130.9, 126.3 (d, *J* 3.1 Hz), 121.4 (dd, *J* 4.6 Hz, *J* 11.1 Hz), 103.6, 75.7, 68.7, 68.1, 63.2, 56.8, 47.4 (d, 5.7 Hz), 39.2. <sup>31</sup>P NMR (CD3OD, 162 MHz): <sup>−</sup>1.0. HRMS calcd for C20H26PNO8+H<sup>+</sup> (M+H)+: 440.1474, found: 440.1470.

#### *3.13. Methyl 2,3-Di-O-Acetyl-*β*-*d*-Gulopyranoside* **25**

Compound **24** (300 mg, 0.82 mmol) was dissolved in 80% aqueous AcOH (5 mL) and the solution was stirred at 80 ◦C for 2 h. When the TLC (TLC, heptane/EtOAc, 1:2, R<sup>f</sup> 0.39) showed complete consumption of the starting material, the solvents were evaporated under reduced pressure and co-evaporated twice with toluene (10 mL). Then, the crude was purified via flash chromatography (Heptane/EtOAc, 3:1–1:2) to obtain pure compound **25** (191 mg, 0.69 mmol, 84%) as a white foam. [α] 25 D <sup>−</sup>30.4 (c 0.8, CHCl3). <sup>1</sup>H NMR (CDCl3, 400 MHz): 5.35 (t, 1H, *<sup>J</sup>*2,3 3.6 Hz, H-3), 5.10 (dd, 1H, *<sup>J</sup>*1,2 8.0 Hz, *J*2,3 3.6 Hz, H-2), 4.68 (d, 1H, *J* 8.0 Hz, H-1), 3.93–3.89 (m, 4H, H4, H-5, H-6a, H-6b), 3.52 (s, 3H, OC*H*3), 2.11 (s, 3H, COC*H*3), 2.03 (s, 3H, COC*H*3). <sup>13</sup>C NMR (CDCl3, 100 MHz): 170.0, 169.9, 99.9, 73.3, 70.6, 69.0, 68.4, 62.8, 56.9, 20.98, 20.95. HRMS calcd for C11H18O8+Na<sup>+</sup> (M+Na)+: 301.0899, found: 301.0898.

#### *3.14. Methyl* β*-*d*-Gulopyranoside* **9**

Compound **25** (120 mg, 0.43 mmol) was dissolved in MeOH (3 mL). NaOMe (1.0 mL, 0.5 M in MeOH) was added and the solution was stirred at room temperature for 2 h (TLC, DCM/MeOH, 5:1, R<sup>f</sup> 0.3). The solution was neutralized with DOWEX 50 W H<sup>+</sup> resin, filtered and the solvents were evaporated under reduced pressure and the crude was purified via flash chromatography (DCM/MeOH, 7:1–3:1) to obtain pure compound **9** (46 mg, 0.23 mmol, 55%) as a colorless oil. [α] 25 D <sup>−</sup>19.2 (c 0.9, CH3OH). <sup>1</sup>H NMR (D2O, 400 MHz): 4.60 (d, 1H, *<sup>J</sup>*1,2 8.4 Hz, H-1), 4.05 (t, 1H, *<sup>J</sup>*3,4 3.6 Hz, H-4), 4.00–3.96 (m, 1H, H-5), 3.80 (dd, 1H, *J*3,4 3.6 Hz, *J*4,5 1.2 Hz, H-4), 3.75 (dd, 1H, *J*6a,6b 12.0 Hz, *J*5,6a 6.4 Hz, H-6a), 3.76 (dd, 1H, *J*6a,6b 12.0 Hz, *J*5,6a 4.8 Hz, H-6b), 3.76 (dd, 1H, *J*1,2 8.4 Hz, *J*2,3 3.6 Hz, H-2), 3.56 (s, 3H, OC*H*3). <sup>13</sup>C NMR (D2O, 100 MHz): 101.5, 73.8, 71.1, 69.3, 68.0, 61.0, 56.9. HRMS calcd for C7H14O6+Na<sup>+</sup> (M+Na)+: 217.0688, found: 217.0687.

#### *3.15. Methyl 3-Azido-2,4,6-Tri-O-Benzoyl-3-Deoxy-*β*-*d*-Gulopyranoside* **29**

Triflic anhydride (235 µL, 1.4 mmol) was added dropwise to a stirred solution of **26** (400 mg, 1.4 mmol) in DCM (10 mL) and pyridine (451 µL, 5.6 mmol) at −30 ◦C and under N<sup>2</sup> atmosphere after which the reaction was allowed to reach rt under 2 h. BzCl (179 µL, 1.54 mmol) was added and the reaction was stirred for another 2 h before the reaction was diluted with DCM (25 mL) and washed with saturated NaHCO<sup>3</sup> (2 × 25 mL). The combined aqueous phases were extracted with DCM (40 mL). The pooled organic phases were dried over MgSO<sup>4</sup> and concentrated to give crude **27**. Sodium azide (637 mg, 9.8 mmol) was added to the crude **27** (≤1.4 mmol) in DMF (15 mL) and the reaction was stirred overnight at 70 ◦C under N<sup>2</sup> atmosphere. The reaction was concentrated to give crude **28**, which was dissolved in 90% AcOH (20 mL) and heated at 80 ◦C for 3 h. The solvent was evaporated in vacuo and co-evaporated with toluene to remove the residual AcOH. The residue was dissolved in pyridine (15 mL), into the solution catalytic amount of DMAP and benzoyl chloride (488 µL, 4.2 mmol) was added subsequently. The solution was stirred at room temperature for 4 h when TLC (heptane/EtOAc, 4:1, R<sup>f</sup> 0.48) showed complete conversion of the starting material to a faster moving spot. The solvents were evaporated in vacuo and co-evaporated with toluene to remove residual pyridine. The solid residue thus obtained was dissolved in EtOAc (50 mL) and washed with 1 N HCl (50 mL), followed by saturated NaHCO<sup>3</sup> and brine (50 mL). The organic layer was collected, dried (Na2SO4), filtered and evaporated in vacuo. The crude was purified by flash chromatography using heptane/EtOAc (6:1 to 5:2) as the eluent to afford pure compound **29** (324 mg, 0.61 mmol, 43% over four steps) as a white amorphous mass. [α] 25 <sup>D</sup> <sup>−</sup>45.3 (c 0.7, CHCl3). <sup>1</sup>H NMR (CDCl3, 400 MHz): 8.14–7.39 (m, 15H, Ar*H*),

5.51 (dd, 1H, *J*1,2 7.6 Hz, *J*2,3 4.0 Hz, H-2), 5.41 (dd, 1H, *J*3,4 4.0 Hz, *J*3,4 0.8 Hz, H-4), 5.00 (d, 1H, *J* 7.6 Hz, H-1), 4.66–4.61 (m, 1H, H-5), 4.52–4.45 (m, 3H, H-3, H-6a, H-6b), 3.58 (s, 3H, OC*H*3). <sup>13</sup>C NMR (CDCl3, 100 MHz): 166.0, 165.3, 165.2, 133.8, 133.6, 133.2, 130.02, 129.98, 129.7, 129.5, 129.0, 128.7, 128.6, 128.5, 128.4, 99.6, 70.3, 70.1, 69.5, 62.4, 60.1, 57.0. HRMS calcd for C28H25N3O8+NH<sup>4</sup> <sup>+</sup> (M+NH4) <sup>+</sup>: 549.1985, found: 549.1989.

#### *3.16. Methyl 3-Amino-2,4,6-Tri-O-Benzoyl-3-Deoxy-*β*-*d*-Gulopyranoside* **30**

A solution of **29** (201 mg, 0.3784 mmol) in MeOH (7 mL) was stirred with Pd(OH)2/C (10% wt., 1 mg per 5 mg of crude, 40 mg) under hydrogen atmosphere at room temperature for 2 h. After the completion of the reaction (as indicated by TLC, heptane/EtOAc, 1:1, R<sup>f</sup> 0.26), the reaction mixture was filtered through a Celite bed and washed with methanol. The filtrate was concentrated under reduced pressure and purified through flash column (heptane/EtOAc, 4:1–1:1) to get the desired compound as a white amorphous solid. Yield: 126 mg (0.2494 mmol, 66%). [α] 25 <sup>D</sup> <sup>−</sup>39.9 (c 0.8, CHCl3). <sup>1</sup>H NMR (CDCl3, 400 MHz): 8.13–7.38 (m, 15H, Ar*H*), 5.38 (dd, 1H, *J*1,2 7.2 Hz, *J*2,3 4.0 Hz, H-2), 5.29 (dd, 1H, *J*3,4 4.4 Hz, *J*4,5 2.4 Hz, H-4), 5.11 (d, 1H, *J* 7.2 Hz, H-1), 4.83–4.79 (m, 1H, H-5), 4.64 (dd, 1H, dd, 1H, *J*6a,6b 11.2 Hz, *J*5,6a 6.8 Hz, H-6a), 4.51 (dd, 1H, dd, 1H, *J*6a,6b 11.2 Hz, *J*5,6b 6.0 Hz, H-6b), 3.90 (t, 1H, *J*3,4, *J*2,3 4.0 Hz, H-3), 3.57 (s, 3H, OC*H*3), 1.97 (bs, 1H, N*H*2). <sup>13</sup>C NMR (CDCl3, 100 MHz): 166.3, 165.9, 165.3, 133.7, 133.5, 133.2, 130.1, 129.9, 129.8, 128.69, 128.67, 128.5, 99.3, 72.1, 71.7, 70.2, 63.3, 57.1, 50.6. HRMS calcd for C28H27NO8+H<sup>+</sup> (M+H)+: 506.1815, found: 506.1817.

#### *3.17. Methyl 3-Benzamido-2,4,6-Tri-O-Benzoyl-3-Deoxy-*β*-*d*-Gulopyranoside* **31**

Compound **30** was dissolved in pyridine (3 mL), into the solution catalytic amount of DMAP and benzoyl chloride (29 µL, 0.2464 mmol) was added subsequently. The solution was stirred at room temperature for 3 h when TLC (heptane/EtOAc, 1:1, R<sup>f</sup> 4.8) showed complete conversion of the starting material to a faster moving spot. The solvents were evaporated in vacuo and co-evaporated with toluene to remove residual pyridine. The solid residue thus obtained was dissolved in EtOAc (7 mL) and washed with 1 (N) HCl (5 mL), followed by saturated NaHCO<sup>3</sup> and brine (5 mL). The organic layer was collected, dried over Na2SO4, filtered and evaporated in vacuo. The crude was purified by flash chromatography using heptane/EtOAc (7:1 to 3:1) as the eluent to afford pure compound **31** (77 mg, 0.1265 mmol, 77%) as a white amorphous solid. [α] 25 <sup>D</sup> <sup>−</sup>48.8 (c 0.6, CHCl3). <sup>1</sup>H NMR (CDCl3, 400 MHz): 8.11–7.29 (m, 20H, Ar*H*), 6.60 (d, 1H, *J*3,N*H*COPh 8.4 Hz, N*H*COPh), 5.96 (dd, 1H, *J* 10.8 Hz, *J* 6.0 Hz, H-4), 5.55 (t, 1H, *J* 2.8 Hz, H-2), 5.34–5.29 (m, 1H, H-3), 4.99 (d, 1H, *J*1,2 2.8 Hz, H-1), 4.92 (dd, 1H, *J*6a,6b 11.6 Hz, *J*5,6a 5.6 Hz, H-6a), 4.86 (dd, 1H, *J*6a,6b 11.6 Hz, *J*5,6b 6.4 Hz, H-6a), 4.77 (dd, 1H, *J*6a,6b 12.4 Hz, *J*4,5 6.0 Hz, H-5), 3.61 (s, 3H, OC*H*3). <sup>13</sup>C NMR (CDCl3, 100 MHz): 167.4, 166.8, 166.2, 165.5, 133.9, 133.8, 133.7, 133.0, 131.7, 130.0, 129.9, 129.7, 129.6, 129.1, 128.7, 128.59, 128.57, 128.3, 127.0, 99.5, 72.4, 71.8, 68.4, 64.5, 60.4, 56.8, 46.3. HRMS calcd for C35H31NO9+H<sup>+</sup> (M+H)+: 610.2077, found: 610.2081.

#### *3.18. Methyl 3-Benzamido-3-Deoxy-*β*-*d*-Gulopyranoside* **10**

Compound **31** (54 mg, 0.0886 mmol) was dissolved in MeOH (2 mL). NaOMe (0.5 mL, 0.5 M in MeOH) was added and the solution was stirred at room temperature for 12 h (TLC, DCM/MeOH, 10:1, R<sup>f</sup> 0.4). The solution was neutralized with DOWEX 50 W H+ resin, filtered and the solvents were evaporated under reduced pressure and the residue was purified by a short flash column using DCM–MeOH (9:1) to afford the compound **10** (19.2 mg, 0.0646 mmol, 73%). [α] 25 <sup>D</sup> −18.3 (c 0.6, CH3OH). <sup>1</sup>H NMR (CD3OD, 400 MHz): 7.83 (d, 2H, *J* 7.6 Hz, Ar*H*), 7.57–7.44 (m, 3H, Ar*H*), 4.70 (d, 1H, *J*1,2 8.4 Hz, H-1), 4.48–4.44 (m, 1H, H-3), 4.01 (dd, 1H, *J*3,4 3.6 Hz, *J*4,5 1.2 Hz, H-4), 3.94 (dd, 1H, *J*1,2 7.6 Hz, *J*2,3 5.2 Hz, H-2), 3.84 (td, 1H, *J*5,6a, *J*5,6a 6.0 Hz, *J*4,5 1.6 Hz, H-5), 3.77 (dd, 1H, *J*6a,6b 11.2 Hz, *J*5,6a 6.0 Hz, H-6a), 3.74 (dd, 1H, *J*6a,6b 11.2 Hz, *J*5,6a 6.0 Hz, H-6b), 3.57 (s, 3H, OC*H*3). <sup>13</sup>C NMR (CD3OD, 100 MHz): 171.4, 164.6, 135.9, 132.7, 129.5, 128.6, 103.4, 75.7, 68.8, 67.8, 62.6, 56.9, 56.0. HRMS calcd for C14H19NO6+H<sup>+</sup> (M+H)+: 298.1291, found: 298.1289.

### *3.19. Expression Constructs, Expression, and Purification of Recombinant Galectins*

Human galectin-1 [25], galectin-2 [26], galectin-3 [27], galectin-4N [19], galectin-4C [19], galectin-8N [28], galectin-8C [28], galectin-9N [29], and galectin-9C [30], were expressed and purified as described earlier. Human galectin-7 was expressed using a pET3c plasmid in *E. coli* BL21-star. The plasmid containing expression optimized DNA encoding the full human galectin-7 sequence (NCBI Reference Sequence: NP\_002298.1) was obtained from GenScript (Piscataway, NJ, USA). Bacterial culture and induction, and galectin purification was essential as described for galectin-3 expressed with the same vector [27]; a typical yield was 1.5–2 mg/L culture. Lactose was removed by chromatography on a PD-10 column (Amersham Biosciences) with repeated ultrafiltration with Centriprep (Amicon).

#### *3.20. Fluorescence Polarization Assay*

Fluorescence polarization experiments were carried out either with a POLARStar plate reader and FLUOstar Galaxy software or with a PheraStarFS plate reader and PHERAstar Mars version 2.10 R3 software (BMG, Offenburg, Germany). The dissociation constant (Kd) values were determined in PBS as described earlier [18,19]. Specific conditions for galectin-1, 2, 3, 4N, 4C, 8N, 8C, 9N, and 9C were kept as reported [29]. Experiments were performed at room temperature with human galectin-7 at 5 µM and the fluorescent probe β-d-galactopyranosyl-(1–4)-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1–3)-β-dgalactopyranosyl-(1–4)-(*N*1-fluorescein-5-yl-carbonylaminomethylcarbonyl)-β-d-glucopyranosylamine [29] at 0.02 µM. All the compounds in Table 1 except 32 were dissolved in a neat DMSO at 100 mM and diluted in PBS to three to six different concentrations to be tested in duplicate. Average K<sup>d</sup> values and SEMs were calculated from 2–8 single-triple point measurements showing between 30%–70% inhibition.

#### **4. Conclusions**

In summary, we report the synthesis and discovery of 3-*C*-methyl-guloside derivatives as highly selective galectin-1 inhibitors with 3-*C*-benzamidomethyl-3-deoxy-gulosides being the most selective structural class. The reason for the exceptional galectin-1-selectivites discovered remains to be elucidated as molecular modelling failed to provide insight into this and experimental structural studies by X-ray diffraction or nmr spectroscopy are likely necessary. Although the galectin-1 affinities are in the high-µM to low mM range, they are significantly higher affinity than that of simple galactosides, such as methyl β-d-galactopyranoside, and thus points towards a novel structural class and synthetic route towards the discovery of galectin-1 inhibitors with high selectivity. This is important in light of the roles of galectin-1 in tumor progression and immune regulation [31,32].

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/15/ 3786/s1.

**Author Contributions:** K.B.P. and M.M. contributed equally to the synthesis and characterization of all compounds. K.B.P wrote the major part of the manuscript. H.L. supervised and analyzed the result of the fluorescence polarization assay. U.J.N. conceived the study, analyzed the data and co-wrote the paper. The manuscript was written with contributions from all authors. All authors have given consent to the final version of the manuscript.

**Funding:** We thank the Swedish Research Council (Grant No. 621-2016-03667), a project grant awarded by the Knut and Alice Wallenberg Foundation (KAW 2013.0022), and Galecto Biotech AB, Sweden for financial support.

**Acknowledgments:** We thank Barbro Kahl-Knutson for excellent assistance with determining affinities by fluorescence polarization and Sofia Essén for excellent assistance with hrms and analytical hplc experiments.

**Conflicts of Interest:** H.L. and U.J.N. are shareholders in Galecto Biotech AB, Sweden, a company developing galectin inhibitors.
