Supporting Information

Robust therapeutic potential of carbazole‒triazine hybrids as a new class of urease

inhibitors: A distinctive combination of nitrogen‒containing heterocycles

Aliya Ibrar,a,† Madiha Kazmi,b,c,† Ajmal Khan,d Sobia Ahsan Halim,d Aamer Saeed,b,* Saifullah

Mehsud,a Ahmed Al-Harrasi,d,* Imtiaz Khane,f,*

aDepartment of Chemistry, Abbottabad University of Science and Technology, Havelian,

Abbottabad, Pakistan

bDepartment of Chemistry, Quaid‒i‒Azam University, Islamabad 45320, Pakistan

cDepartment of Chemistry, University of Gujrat, Rawalpindi Sub‒campus, Satellite Town,

Rawalpindi, Pakistan

dNatural and Medical Sciences Research Center, University of Nizwa, P.O Box 33, Postal Code

616, Birkat Al Mauz, Nizwa, Sultanate of Oman

eSchool of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL,

United Kingdom

fManchester Institute of Biotechnology, The University of Manchester, 131 Princess Street,

Manchester M1 7DN, United Kingdom

†These authors contributed equally to this work.

Contents

Figure S1a. Superimposed view of the docked conformation of β-mercaptoethanol (shown in

green sticks) on its X-ray conformation (white sticks) in the binding site of 1UBP.

Figure S1b. Superimposed view of the docked conformation of diamidophosphate on its X-

ray conformation in the binding site of 3UBP.

Figure S1c. Superimposed view of the docked conformation of acetohydroxamic acid (green

sticks) on its cognate conformation (white sticks) in the active site of 4UBP.

Figure S1d. Superimposed view of the docked conformation of citrate (green sticks) on its

cognate conformation (white sticks) in the active site of 4AC4.

Table S1. Conformational sampling/cluster analysis of docked conformations of compounds

3a-3j (3h was excluded from this study).

Figure S2a. 2D presentation of protein-ligand interactions of 3a in the active site of urease.

Figure S2b. 2D presentation of protein-ligand interactions of 3b in the active site of urease.

Figure S2c. 2D presentation of protein-ligand interactions of 3c in the active site of urease.

Figure S2d. 2D presentation of protein-ligand interactions of 3d in the active site of urease.

Figure S2e. 2D presentation of protein-ligand interactions of 3e in the active site of urease.

Figure S2f. 2D presentation of protein-ligand interactions of 3f in the active site of urease.

Figure S2g. 2D presentation of protein-ligand interactions of 3g in the active site of urease.

Figure S2h. 2D presentation of protein-ligand interactions of 3i in the active site of urease.

Figure S2i. 2D presentation of protein-ligand interactions of 3j in the active site of urease.

Figure S1a. Superimposed view of the docked conformation of β-mercaptoethanol (shown in

green sticks) on its X-ray conformation (white sticks) in the binding site of 1UBP.

Figure S1b. Superimposed view of the docked conformation of diamidophosphate on its X-ray

conformation in the binding site of 3UBP.

Figure S1c. Superimposed view of the docked conformation of acetohydroxamic acid (green

sticks) on its cognate conformation (white sticks) in the active site of 4UBP.

Figure S1d. Superimposed view of the docked conformation of citrate (green sticks) on its

cognate conformation (white sticks) in the active site of 4AC4.

Table S1. Conformational sampling/cluster analysis of docked conformations of compounds 3a-3j (3h was excluded from this study)Entry Molecule Score Interactions 1 3a.cdx -9.8206 R (Gly280), D (Arg339), a (Ala366) 2 3a.cdx -9.66199 D (Arg339), a (Ala366) 3 3a.cdx -9.55986 D (Arg339), OO (Wat2218) 4 3a.cdx -9.23428 a (Ala366), O (Wat2261) 5 3a.cdx -9.08858 R (Gly280, Cys322, His323) 6 3a.cdx -8.71562 RR (Gly280, (His323) 7 3a.cdx -8.48867 R (His323) 8 3a.cdx -7.77158 RR (Gly280, Ala170, Cys322) 9 3a.cdx -7.67355 R (His249) 10 3a.cdx -7.66535 R (Gly280), a (Ala366) 11 3a.cdx -7.63119 R (Arg339) 12 3a.cdx -7.58389 AA (His323) 13 3a.cdx -7.42725 R (His249, Arg339), A (Asp363) 14 3a.cdx -6.92009 R (His222), O (Wat2339) 15 3a.cdx -6.57655 D (Arg339) 16 3a.cdx -6.41366 No Interactions 17 3a.cdx -6.36813 R (His222, Arg339), O (Wat2339) 18 3a.cdx -6.34948 D (Arg339) 19 3a.cdx -6.26091 R (His323) 20 3a.cdx -6.0895 RR (His323), A (Asp363) 21 3a.cdx -5.74123 R (Cys322, Arg339) 22 3a.cdx -5.47061 D (Arg339) 23 3a.cdx -5.25413 D (Arg339) 24 3a.cdx -5.2106 No Interactions 25 3b.cdx -10.1705 R (Gly280), 26 3b.cdx -9.96179 R (Ala366) 27 3b.cdx -9.67852 a (Ala366), O (Wat2261) 28 3b.cdx -9.44122 R (Gly280), O (Wat2261) 29 3b.cdx -9.41261 R (Gly280), O (Wat2218) 30 3b.cdx -8.98286 R (Gly280) 31 3b.cdx -8.96895 R (His323) 32 3b.cdx -8.25761 No Interactions 33 3b.cdx -7.57474 D (Arg339) 34 3b.cdx -7.53563 No Interactions 35 3b.cdx -7.50116 No Interactions 36 3b.cdx -7.44619 R (Ala170, His222, His323) 37 3b.cdx -7.35893 A (Asp224), a (Gly280) 38 3b.cdx -7.18202 R (Wat2218) 39 3b.cdx -7.07184 D (Arg339), R (His323)

40 3b.cdx -6.96908 D (His249) 41 3b.cdx -6.80981 D (Arg339) 42 3b.cdx -6.49966 A (His249), R (Gly280, Cys322) 43 3b.cdx -6.48742 D (His323) 44 3b.cdx -6.38948 R (His323) 45 3b.cdx -6.28045 R (Arg339) 46 3b.cdx -5.76542 D (Arg339) 47 3b.cdx -5.08843 D (Arg339) 48 3b.cdx -4.73702 R (Cys322), A (His323) 49 3b.cdx -4.73172 R (Cys322), AA (His323), a (Ala366) 50 3c.cdx -9.97975 O (Wat2261) 51 3c.cdx -9.96734 O (Wat2261) 52 3c.cdx -9.79537 R (His222, His323) 53 3c.cdx -9.73493 R (Gly280) 54 3c.cdx -9.73282 R (His222, Gly280, His323) 55 3c.cdx -8.48048 D (Arg339) 56 3c.cdx -8.19608 R (His222), D (Cys322), O (Wat2339) 57 3c.cdx -8.01476 R (His222, Cys322, His324) 58 3c.cdx -7.68385 R (His222), AA (His323) 59 3c.cdx -7.16473 D (Met367) 60 3c.cdx -7.01971 AA (Arg339) 61 3c.cdx -6.99809 R (His324), OO (Wat2258) 62 3c.cdx -6.97085 No Interactions 63 3c.cdx -6.84959 D (Asp224), R (Wat2218) 64 3c.cdx -6.69662 R (His249), DD (Asp363) 65 3c.cdx -6.68878 R (Gly280), AA (Arg339) 66 3c.cdx -6.48082 R (His323) 67 3c.cdx -6.116 AA (Arg339) 68 3c.cdx -5.05657 No Interactions 69 3c.cdx -4.8331 D (Thr252), AA (His323), 70 3c.cdx -4.69959 D (Thr252), AA (His323) 71 3d.cdx -11.1795 RR (His323) 72 3d.cdx -10.034 RR (His323), O (Wat2261) 73 3d.cdx -9.75145 R (Gly280), O (Wat2261) 74 3d.cdx -9.39031 R (His222, Gly280, His323) 75 3d.cdx -9.26057 R (His323) 76 3d.cdx -9.03558 d (Ala366) 77 3d.cdx -8.65224 O (Wat2218) 78 3d.cdx -8.3374 R (His222), D (Thr252), R (Cys322, His324) 79 3d.cdx -7.81044 RR (Wat2218) 80 3d.cdx -7.75054 No Interactions 81 3d.cdx -7.3918 R (Gly280, His323), dd (Ala366)

82 3d.cdx -7.29066 R (Cys322) 83 3d.cdx -7.04795 AA (Arg339) 84 3d.cdx -6.60575 R (Cys322), O (Wat2339) 85 3d.cdx -6.54712 R (His323), AA (Arg339) 86 3d.cdx -6.38312 R (Arg339) 87 3d.cdx -6.33299 RR (His324), D (Asp363) 88 3d.cdx -4.53135 R (Cys322), AA (His323) 89 3e.cdx -10.0929 R (Gly280, His323, His222, Ala170), AA (Arg339), dd (Ala366) 90 3e.cdx -9.69481 dd (Ala366), O (Wat2261) 91 3e.cdx -9.30768 R (His323) 92 3e.cdx -9.03554 dd (Ala366), O (Wat2261) 93 3e.cdx -7.81781 R (His222), R (Cys322), RR (His324), O (Wat2339) 94 3e.cdx -7.6706 RR (Wat2218) 95 3e.cdx -7.56134 R (His324) 96 3e.cdx -7.35843 R (Cys322), AA (Arg339) 97 3e.cdx -7.20409 No Interactions 98 3e.cdx -7.11347 AA (Arg339) 99 3e.cdx -7.08546 D (Glu166) 100 3e.cdx -6.79258 D (Glu166), AA (His323) 101 3e.cdx -6.47946 R (His323) 102 3e.cdx -6.37832 R (Arg339) 103 3e.cdx -5.58103 D (Glu223), d (Gly280), R (His324) 104 3e.cdx -5.50528 D (Thr252) 105 3e.cdx -4.41777 D (Cys322), R (Arg339, Wat2218)) 106 3e.cdx -3.2681 D (Glu166, Asp224, Cys322) 107 3e.cdx 54.7498 D (Glu223) 108 3f.cdx -10.4578 dd (Ala366), AA (Arg339), R (Ala170, Gly280, His222, His323,) 109 3f.cdx -10.2627 dd (Ala366), O (Wat2261) 110 3f.cdx -9.70949 d (Ala366) 111 3f.cdx -8.84433 AA (Lys169), O (Wat2218) 112 3f.cdx -8.24397 RR (Wat2218) 113 3f.cdx -8.23879 R (His222), A (His323) 114 3f.cdx -8.12539 R (His324), A (Arg339), O (Wat2261) 115 3f.cdx -7.90068 AA (Lys169), R (His222), R(Cys322) 116 3f.cdx -7.79443 A (Lys169), R (Cys322), O (Wat2218, Wat2339) 117 3f.cdx -7.71833 AA (Arg339) 118 3f.cdx -7.70257 R (Cys322), AA (Arg339) 119 3f.cdx -7.65185 R (Gly280), A(His324), AA (Arg339) 120 3f.cdx -7.41349 AA (Arg339) 121 3f.cdx -7.39378 AA (Lys169)

122 3f.cdx -6.82267 AA(Lys169), R (His323) 123 3f.cdx -6.53545 R(His323), dd (Ala366) 124 3f.cdx -6.5039 AA (Arg339) 125 3f.cdx -6.40607 A (Lys169), O (Wat2258) 126 3f.cdx -6.14669 AA (Lys169) 127 3f.cdx -6.10857 ---- 128 3f.cdx -5.73509 AA (Lys169, His323), d (Ala366) 129 3f.cdx -4.55096 A (His249, His324), AA (His323) 130 3g.cdx -9.86672 R (Gly280), AA (Arg339), dd (Ala366) 131 3g.cdx -9.57746 AA (Arg339), O (Wat2218) 132 3g.cdx -9.57414 dd (Ala366), O (Wat2261) 133 3g.cdx -9.32295 R (Gly280), dd (Ala366), O (Wat2261) 134 3g.cdx -9.16659 R (Gly280) 135 3g.cdx -8.92034 R (His222), D (Cys322), O (Wat2339) 136 3g.cdx -8.32138 R (Lys169) 137 3g.cdx -8.15552 No Interactions 138 3g.cdx -8.14653 No Interactions 139 3g.cdx -7.99569 R (His222) 140 3g.cdx -7.8619 No Interactions 141 3g.cdx -7.16522 AA (Met367) 142 3g.cdx -7.08089 No Interactions 143 3g.cdx -7.05247 No Interactions 144 3g.cdx -6.99508 RR (Arg339), O (Wat2339) 145 3g.cdx -6.97557 R (Gly280), AA (Arg339) 146 3g.cdx -6.69926 AA (Arg339) 147 3g.cdx -6.132 R (His323) 148 3g.cdx -5.95047 A (Arg339) 149 3g.cdx -5.64377 No Interactions 150 3g.cdx -5.11726 R (Cys322), AA (His323) 151 3i.cdx -10.2823 R (Lys169) 152 3i.cdx -10.031 D (Cys322) 153 3i.cdx -10.0203 R (His324) 154 3i.cdx -9.88658 R (His323) O (Wat2218) 155 3i.cdx -9.81559 D (Arg339), R (His222, His323) 156 3i.cdx -9.34405 R (His323) 157 3i.cdx -8.54138 dR (His323) 158 3i.cdx -8.41511 No Interactions 159 3i.cdx -8.35938 R (His222, His324) 160 3i.cdx -8.33657 R (His222, Cys322, His324) 161 3i.cdx -8.24327 R (Cys322), A (His324) 162 3i.cdx -8.21263 R (His324), D (Met367) 163 3i.cdx -8.21204 R (His222, Cys322)

164 3i.cdx -8.20651 R (Cys322) 165 3i.cdx -8.1593 No Interactions 166 3i.cdx -8.10823 R (His323), R (Wat2218) 167 3i.cdx -8.01769 R (His323), D (Met367) 168 3i.cdx -8.00116 R (His323), D (Met367) 169 3i.cdx -7.9787 D (Asp224), RR (His324), O (Wat2258) 170 3i.cdx -7.93682 No Interactions 171 3i.cdx -7.89052 RR (His324), D (Met367) 172 3i.cdx -7.83811 R (His222), D (Cys322) 173 3i.cdx -7.47031 D (Met367) 174 3i.cdx -7.34728 R (His324), D (Met367) 175 3i.cdx -7.29557 No Interactions 176 3i.cdx -6.2126 R (His323), D (Met367) 177 3j.cdx -11.7547 dR (His323), dd (Ala366), O (Wat2261) 178 3j.cdx -11.5768 R (Gly280), dR (His323), dd (Ala366), O (Wat2261) 179 3j.cdx -10.5355 R (His323) 180 3j.cdx -10.4205 R (His324), D (Asp332), O (Wat2258) 181 3j.cdx -10.3334 R (His324) 182 3j.cdx -10.1449 R (His324) 183 3j.cdx -10.1086 R (Cys322) 184 3j.cdx -9.97459 R (His323), O (Wat2258) 185 3j.cdx -9.63248 R (His323), R(His324) 186 3j.cdx -9.61175 No Interactions 187 3j.cdx -9.54414 R (His324), O (Wat2258) 188 3j.cdx -9.53527 R (Cys322), R (His323) 189 3j.cdx -9.43898 R (His222), d (His323) 190 3j.cdx -9.2527 D (Met367), O (Wat2339) 191 3j.cdx -9.1132 O (Wat2258) 192 3j.cdx -9.09546 No Interactions 193 3j.cdx -8.96058 No Interactions 194 3j.cdx -8.91659 D (Asp224), R (His324) 195 3j.cdx -8.87503 No Interactions 196 3j.cdx -8.8387 D (Asp332) 197 3j.cdx -8.52384 R (His324) 198 3j.cdx -8.18223 R (His323, Asp224), D (Arg339) 199 3j.cdx -8.17869 R (His324) 200 3j.cdx -8.1629 R (His324) 201 3j.cdx -8.06175 D (Asp224) 202 3j.cdx -7.8548 No Interactions 203 3j.cdx -7.5386 No Interactions 204 3j.cdx -7.09849 R (His324)

205 3j.cdx -6.468 R (Wat2220) 206 3j.cdx -6.20126 A (Lys169), D (Cys322)

D = Side chain hydrogen bond donor; A = Side chain hydrogen bond acceptor; d = Backbone hydrogen bond donor; a = Backbone hydrogen bond acceptor; O = Solvent hydrogen bond; I = Ionic interactions; R = Hydrophobic interactions (π-π, π-H, π-cation).The selected conformation (based on docking score and interaction) are highlighted in green color in Table S1.

Figure S2a. 2D presentation of protein-ligand interactions of 3a in the active site of urease.

Figure S2b. 2D presentation of protein-ligand interactions of 3b in the active site of urease.

Figure S2c. 2D presentation of protein-ligand interactions of 3c in the active site of urease.

Figure S2d. 2D presentation of protein-ligand interactions of 3d in the active site of urease.

Figure S2e. 2D presentation of protein-ligand interactions of 3e in the active site of urease.

Figure S2f. 2D presentation of protein-ligand interactions of 3f in the active site of urease.

Figure S2g. 2D presentation of protein-ligand interactions of 3g in the active site of urease.

Figure S2h. 2D presentation of protein-ligand interactions of 3i in the active site of urease.

Figure S2i. 2D presentation of protein-ligand interactions of 3j in the active site of urease.