**2. Results**

Compound **1** was obtained as a colorless bulk crystal. Its molecular formula C11H12BrN5O3 was determined by HRESIMS with the ion peak at *m*/*z* 342.0197, and 344.0170 with the proportion of 1:1 (calcd. for [M + H]+ *m*/*z* 342.0196, and 344.0176), with the unsaturation degree of 8. The 13C NMR and DEPT spectra revealed 11 resonances (Table S1) including one methylene, three methines, and seven non-protonated carbons. The chemical shifts ranging from *δ*C 104.3 to 133.6 ppm showed six olefinic carbons, which included two methines and four non-protonated carbons. In the low field of the 13C NMR spectrum with the chemical shifts of *δ*C 170.5, 162.1, and 156.4, there could be two carbonyl groups and one guanidyl group that exist in this molecule. The 1H NMR and HSQC spectra (Table S2) showed the presence of two olefinic protons at *δ*H 6.36 (1H, d, *J* = 2.3 Hz), and *δ*H 6.04 (1H, t, *J* = 6.8 Hz), one methine proton at *δ*H 5.21 (1H, d, *J* = 8.5 Hz), and one methylene proton at *δ*H 3.42 (2H, m), and there were three additional heteroatomic protons at *δ*H 12.71 (brs), *δ*H 7.80 (1H, d, *J* = 8.3 Hz), and *δ*H 7.79 (1H, q, *J* = 4.7 Hz). The 1H-1H COSY spectrum (Figure 2) revealed a pyrrole conjugate ring system and another spin system with the correlations between 1-NH and H-3, between 15-NH and H-11, between 7-NH and H2-8, and between H2-8 and H-9. Key HMBC correlations (Figure 2) of 1-NH/C-3, C-4, and C-5, H-3/C-2, and C-4, 7-NH/C-5, and C-9, H2-8/C-6, C-9, and C-10, H-9/C-4, C-8, and C-10 determined the existence of a 5/7 bicyclic 2-bromo-6,7-dihydropyrrolo [2,3-*c*] azepin-8(1*H*)-one skeleton (Figure 2). The already existential bicyclic skeleton and four double bonds occupied six degrees of unsaturation, and the remaining two degrees pointed out that there was no other ring systems in compound **1**. The final planar structure of compound **1** was settled down by the HMBC correlations from H-11 to C-4, C-9, C-10, C-12, and C-14, together with the correlations from H-9 to C-11 as well as 15-NH to C-12. A suitable bulk single crystal of compound **1** was obtained to perform the X-ray diffraction experiment, which ensured the planar structure of **1** (Figure 3). The space group of **1** indicated that it was a scalemic mixture, and ECD calculation finally determined the absolute configurations of **1a** and **1b** isolated through chiral HPLC by comparison with each of their ECD spectra (Figures S1 and S2).

**Figure 2.** The key COSY (bolds), and HMBC (arrows) correlations of **1**–**12**.

**Figure 3.** The X-ray structures of compounds **1**, **5**, and **10**.

Compound **2** was obtained as a yellow oil and its molecular formula was determined as C11H11N5O3 by the [M + H]+ ion peak presented at *m*/*z* 262.0936 (calcd. C11H12N5O3 for *m*/*z* 262.0935) in the HRESIMS spectrum with nine unsaturation degrees. One more unsaturation degree and the similar chemical shifts of the carbons (Table S1) with **1** indicated that compound **2** may have a 5/7/5 tricyclic spongiacidin-PIA skeleton, close to the known compound **13**. The HMBC correlations (Figure 2) of 1-NH/C-3, and C-4, H-2/C-3, C-4, and C-5, H-3/C-2, C-4, and C-5, 7-NH/C-5, C-8, and C-9, H2-8/C-6, C-9, and C-10, and H-9/C-2, C-4, C-10, and C-11, together with the COSY correlations of 1-NH/H-2/H-3, and 7-NH/H2-8/H-9, constructed the classic 5/7/5 tricyclic structure with the hydroxyl group substituted at C-9, taking the chemical shift of C-9 (*δ*C 62.8) into consideration. Thus, the planar structure of **2** was established.

The 1D and 2D NMR spectra as well as the HRESIMS spectrum indicated that compound **3** possessed the same planar structure with **2** (Figure 2). The *Z*- and *E*-configuration of double bond Δ10,11 in spongiacidin-type PIAs often concomitantly appeared and their differences can be attributed to the anisotropic effect of the carbonyl at C-12 [16]. Two configurations of known compounds **13** and **14** could be distinguished by the chemical shift values of H-3 and H2-9, but the existence of 9-OH substituted in compounds **2** and **3** made it so that the judgement rule did not work [compound 2: *δ*H 6.45 (H-3), 5.80 (H-9); compound **3**: *δ*H 6.43 (H-3), 5.83 (H-9)]. Through careful analysis of their 13C NMR spectra, we found the double bonds of compounds **2** and **13** could be in the *Z*-configuration [16] because the signals for C-4, C-10, C-11, C-12, and C-14 were weak compared with the stronger signals for C-2, C-3, C-5, C-6, C-8, and C-9, while the carbon signals of compounds **3** and **14** were distributed on average comparatively (Figure 4). Known compound **15** co-isolated was also determined to be of a *Z*-configuration with the evidence of its carbon signal analysis in the 13C NMR spectrum. Thus, the double bond of Δ10,11 in compound **2**

was determined as the *Z*-configuration and compound **3** was the *E*-configuration. C-9s absolute configurations of compounds **2a**, **2b** and **3a**, **3b** were all identified based on the ECD calculations together with the chiral HPLC method (Figures S3–S6).

**Figure 4.** The comparison of the 13C NMR spectra of compounds **2**, **3**, **13**, **14**, and **15** (125 MHz, DMSO-*d*6).

Compound **4** is a molecule similar to compounds **2** and **3**, with the same molecular formula C11H11N5O3 by the [M + H]+ ion peak presented at *m*/*z* 262.0931 (calcd. for *m*/*z* C11H12N5O3 262.0935) in the HRESIMS spectrum. The key 1H-1H COSY correlations between 7-NH and H2-8, and between H-11 and 15-NH uncovered the different structure of **4**, and its final planar structure was determined by the HMBC correlations from 1-NH to C-2, C-3, C-4, and C-5, from H-2 to C-3, C-4, and C-5, from H-3 to C-2, C-4, C-5, and C-10, from 7-NH to C-5, C-8, and C-9, from H-8 to C-6, C-9, and C-10, from H-11 to C-4, C-9, C-10, C-12, and C-14, from 15-NH to C-10, and from 9-OH to C-8, C-9, and C-10 (Figure 2). ECD calculation was also carried out to determine the absolute configurations of **4a** and **4b** (Figures S7 and S8).

Compound **5** was obtained as colorless bulk crystals. Its molecular formula was determined to be C12H13N5O2 by HRESIMS (*m*/*z* 260.1148, calcd. [M + H]+ for *m*/*z* 260.1142), which required nine degrees of unsaturation. The 1D and 2D NMR data revealed its similarity with the known compound **13**, with the only difference at 13-NMe with the extra signals of *δ*H 3.11 and *δ*C 25.8 in the 1H and 13C NMR spectra (Tables S1 and S2). The HMBC correlations from 13-NMe to C-12 and C-14 further confirmed the planar structure of **5** (Figure 2). Fortunately, a suitable bulk crystal was obtained followed by X-ray diffraction (Figure 3), and the result showed that the previously proposed rule of distinguishing *Z*/*E* configurations of double bond Δ10,11 in 5/7/5 spongiacidin-type PIAs was trustworthy.

The molecular formula of compound **6**, obtained as a yellow oil, was determined to be C12H14BrN5O3, according to the HRESIMS results, which showed a protonated molecular ion at *m*/*z* 356.0357, 358.0329 (calcd. for [M + H]+, *m*/*z* 356.0353, 358.0332). The analysis of the 1D and 2D NMR spectra (Tables S3 and S4) of **6** indicated that it had a very similar structure with the reported compound (**16**) obtained by organic synthesis [17], with the only difference of 2-OMe rather than 2-OH. The relative configuration of **6** was determined by DP4+ analysis, where the results showed that the only possibility was 2*R*\*6*R*\*10*S*\*, and further ECD calculations confirmed the absolute configuration of **6** was 2*S*6*S*10*R* (Figures S9 and S10).

Compounds **7**–**9** were 5/7 bicyclic pyrrole alkaloids, in which compounds **8** and **9** were 2-bromo substituted ones. Their structures were confirmed by HRESIMS and NMR data (Tables S3 and S4). Compounds **7** and **8** were two pairs of scalemic mixture, and their absolute configurations were solved by the chiral HPLC and ECD calculation method (Figures S11–S14).

Compounds **10**–**12** were simple pyrrole alkaloids with the 2-carboxyl and 3-bromo characteristic, which were pairs of scalemic mixture. Their planar structures were determined by HRESIMS, NMR (Tables S3 and S4), and single crystal X-ray diffraction (Figure 3), and the absolute configurations were confirmed by chiral HPLC and ECD calculations (Figures S15–S20).

Five characteristic skeletons of the alkaloids (**1**–**12**) above-mentioned were isolated from the sponge *Stylissa massa*. Compound **1** was the first identified precursor metabolite of the classic 5/7/5 tricyclic skeleton with unesterified guanidine and carboxyl groups. Through the NMR data analysis of compounds **2**, **3**, **13**, **14**, and **15**, an experience rule to determine the *Z*/*E* configurations of double bond Δ10,11 was summarized based on the signal intensity in the 13C NMR spectra (Figure 4).

Some guanidine compounds were reported to exhibit advantageous biological activities on diabetes [14], which indicated the following aldose reductase (ALR2) assay in vitro. We successfully obtained the protein AKR1B1 (ALR2) by genetic engineering methods (Figure S21) and compounds **1**–**16** were tested. Compounds **2**–**5** and **13**–**15**, representative of 5/7/5 tricyclic spogiacidin-type PIA compounds, displayed superior inhibitory activities compared with other compounds (Figure S22) with epalrestat as the positive control. Further concentration gradient experiments carried out to calculate their IC50 values showed results that ranged from 8.6 to 13.6 μM (Figure S23). The analysis of the structure–activity relationships indicated that 9-OH and 13-NMe may enhance the ALR2 inhibitory activities of this spongiacidin-alkaloid family. Compounds **13** and **14** with the basic 5/7/5 spongiacidin skeleton without stereoscopic configuration were isolated as the major metabolites in sponge *Stylissa massa*. In order to research the interaction mechanism between spongiacidin-skeleton compounds and ALR2, we carried out surface plasmon resonance (SPR) binding assays of compounds **13** and **14** under the Biacore T200 instrument, where the results showed the binding power (KD value) between ALR2 and compound **13** was 12.5 μM (Figure S24), and **14** was 6.9 μM (Figures 5 and S25 ). Molecular docking using the GBVI/WSA ΔG rescoring method was applied to screen the best docking pose between ALR2 and compound **14**. The results showed that the pyrrole and imidazole ring systems were binding to the pocket of ALR2 by H– π and π–π conjugate bonds, respectively (Figure 5).

**Figure 5.** The ALR2 inhibitory activity for compound **14**. ( **A**) Concentration dependent curve of the ALR2 inhibitory assay for **14**. (**B**) The result of the surface plasmon resonance (SPR) binding assay of **14** and ALR2 with a KD value of 6.89 μM. ( **C**) Ligand interactions between ALR2 and **14**. ( **D**) The 3D binding model of compound **14** with ALR2, the surface of the protein is shown in grey, and the interaction bond is shown in the red dotted line.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV and ECD spectra were obtained on a Jasco J-810 spectropolarimeter (Tokyo, Japan). The NMR spectra were measured by a Bruker AVANCE III 500 MHz spectrometer (Bruker company, Fällanden, Switzerland). The 2.50 ppm and 39.5 ppm resonances of DMSO-*d*6 were used as internal references for the 1H and 13C NMR spectra, respectively. HRESIMS data were measured on Micromass Q-Tof Ultima Global GAA076LC (Waters, Milford, CT, USA) and Thermo Scientific LTQ Orbitrap Exploris 480 mass spectrometers (Waltham, MA, USA). X-ray data were obtained by a Rigaku Xtalab Synergy using Cu-K α radiation (Tokyo, Japan). Semi-preparative HPLC utilized an ODS column (Agilent XDB C-18, 9.6 × 250 mm, 5 μm). Silica gel (200–400 mesh, Qingdao, China) was used for column chromatography, precoated silica gel plates (GF254, Qingdao, China) were used for TLC, and spots were visualized by heating SiO2 plates sprayed with 10% H2SO4 in EtOH.

## *3.2. Sponge Material*

The marine sponge *Stylissa massa* was collected from the Xisha Islands of the South China Sea in June 2013, and was frozen immediately after collection. The specimen was identified by Nicole J. de Voogd, National Museum of Natural History, Leiden, The Netherlands. The voucher specimen (No. XS-2013-07) was deposited at lab A1007, School of Pharmacy, Qingdao University, P. R. China.

#### *3.3. Extraction and Isolation*

*Stylissa massa* (8.0 kg, wet weight) was crushed and then extracted with MeOH four times (3 days each time) at room temperature. The combined solutions were concentrated in vacuo and the residue was subsequently desalted to yield the organic extract (191.0 g). The extract was subjected to silica gel vacuum liquid chromatography (VLC), eluting with a gradient of petroleum ether/EtOAc (from 10:1 to 0:1, *v*:*v*) and subsequently CH2Cl2/MeOH (from 10:1 to 0:1, *v*:*v*) to obtain 17 fractions (Fr.1–Fr.17). Fr.5 (0.5 g) was subjected to a silica gel CC (CH2Cl2/MeOH, 20:1, *v*:*v*) to give three fractions Fr.5-1–Fr.5-3. Fr.5-1 (200 mg) was then subjected to a silica gel CC (petroleum ether/EtOAc, from 5:1 to 1:1, *v*:*v*) to give five fractions Fr.5-1-1–Fr.5-1-5. Fr.5-1-2 was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 25:75, *v*/*v*; 2.0 mL/min, 33 min) to afford compound **12** (*<sup>t</sup>*R = 21.8 min, 1.0 mg) and compound **11** (*<sup>t</sup>*R = 24.3 min, 1.0 mg). Fr.7 (3.0 g) was subjected to an ODS CC (MeOH/H2O, from 5:95 to 100:0, *v*:*v*) to give six fractions Fr.7-1–Fr.7-6. Fr.7-2 was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 20:80–60:40, *v*/*v*; 2.0 mL/min, 45 min) to afford compound **10** (*<sup>t</sup>*R = 19.5 min, 3.0 mg). Fr.7- 6 was purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 5:95–100:0, *v*/*v*; 2.0 mL/min, 48 min) to afford compound **8** (*<sup>t</sup>*R = 40.3 min, 5.8 mg) and compound **9** (*<sup>t</sup>*R = 45.0 min, 4.0 mg). Fr.8 (14.5 g) was subjected to a silica gel CC (CH2Cl2/MeOH, 50:1–1:1, *v*:*v*) to give nine fractions Fr.8-1–Fr.8-9. Fr.8-1 (5.0 g) was then subjected to a ODS CC (MeOH/H2O, 50:1–1:1, *v*:*v*) to give five fractions Fr.8-1-1–Fr.8- 1-5. Fr.8-1-3 was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 5:95–100:0, *v*/*v*; 2.0 mL/min, 35 min) to afford compound **16** (*<sup>t</sup>*R = 19.2 min, 2.0 mg), compound **6** (*<sup>t</sup>*R = 23.8 min, 3.7 mg) and compound **7** (*<sup>t</sup>*R = 23.9 min, 3.0 mg). Fr.8-1-5 (4.0 g) was purified subjected to an ODS CC (MeOH/H2O, from 5:95 to 100:0, *v*:*v*) to give six fractions Fr.8-1-5-1–Fr.8-1-5-6. Fr.8-1-5-4 (200 mg) was purified by semipreparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 5:95–100:0, *v*/*v*; 2.0 mL/min, 36 min) to afford compound **3** (*<sup>t</sup>*R = 21.1 min, 2.6 mg), compound **5** (*<sup>t</sup>*R = 26.1 min, 3.7 mg), and compound **13** (*<sup>t</sup>*R = 24.7 min, 200 mg). Fr.15 (17.2 g) was subjected to a silica gel CC (CH2Cl2/MeOH, from 10:1 to 1:1, *v*:*v*) to give four fractions Fr.15-1–Fr.15-4. Fr.15-2 (1.1 g) was then subjected to an ODS CC (MeOH/H2O, from 5:95 to 100:0, *v*:*v*) to give six fractions Fr.15-2-1–Fr.15-2-6. Fr.15-2-4 (300 mg) was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 10:90, *v*/*v*; 2.0 mL/min, 55 min) to afford

compound **2** (*<sup>t</sup>*R = 29.1 min, 16.0 mg), compound **15** (*<sup>t</sup>*R = 44.3 min, 6.0 mg), and compound **14** (*<sup>t</sup>*R = 47.5 min, 150 mg). Fr.17 (20.0 g) was subjected to an ODS CC (MeOH/H2O, from 5:95 to 100:0, *v*:*v*) to give seven fractions Fr.17-1–Fr.17-7. Fr.17-3 (800 mg) was then subjected to a silica gel CC (CH2Cl2/MeOH, from 10:1 to 1:1, *v*:*v*) to give six fractions Fr.17-3-1–Fr.17- 3-6. Fr.17-3-3 was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 5:90–100:0, *v*/*v*; 2.0 mL/min, 40 min) to afford compound **4** (*<sup>t</sup>*R = 18.5 min, 6.8 mg). Fr.17-3-4 was then purified by semi-preparative HPLC (ODS, 5 μm, 250 × 9.6 mm; MeOH/H2O, 5:90–100:0, *v*/*v*; 2.0 mL/min, 38min) to afford compound **1** (*<sup>t</sup>*R = 21.2 min, 5.9 mg).

Compound **1:** Colorless crystals; UV (MeOH) λmax 226 nm; 1H and 13C NMR (DMSO*d*6) data, see Tables S1 and S2; HRESIMS *m*/*z* 342.0184, 344.0162 ([M + H]+ (calcd. for C11 H13BrN5O3, 342.0184, 344.0165); compound **1a**: [α]<sup>20</sup> D −19.7 (c 0.1, MeOH), compound **1b**: [α]<sup>20</sup> D 27.3 (c 0.1, MeOH).

Compound **2:** Yellow oil; UV (MeOH) λmax 354 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S1 and S2; HRESIMS *m*/*z* 262.0936 ([M + H]+ (calcd. for C11 H12 N5O3, 262.0935); compound **2a**: [α]<sup>20</sup> D −40.3 (c 0.1, MeOH), compound **2b**: [α]<sup>20</sup> D 34.5 (c 0.1, MeOH).

Compound **3:** Yellow oil; UV (MeOH) λmax 346 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S1 and S2; HRESIMS *m*/*z* 262.0933 ([M + H]+ (calcd. for C11 H12 N5O3, 262.0935); compound **3a**: [α]<sup>20</sup> D −29.8 (c 0.1, MeOH), compound **3b**: [α]<sup>20</sup> D 36.3 (c 0.1, MeOH).

Compound **4:** Yellow oil; UV (MeOH) λmax 346 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S1 and S2; HRESIMS *m*/*z* 262.0931 ([M + H]+ (calcd. for C11 H12 N5O3, 262.0935); compound **4a**: [α]<sup>20</sup> D 30.1 (c 0.1, MeOH), compound **4b**: [α]<sup>20</sup> D −18.4 (c 0.1, MeOH).

Compound **5:** Colorless crystals; UV (MeOH) λmax 354 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S1 and S2; HRESIMS *m*/*z* 260.1148 ([M + H]+ (calcd. for C12H14N5O2, 260.1142).

Compound **6:** Yellow oil; UV (MeOH) λmax 216 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 356.0357, 358.0353 ([M + H]+ (calcd. for C12 H15BrN5O3, 356.0353, 358.0332); [α]<sup>20</sup> D−19.5 (c 0.1, MeOH).

Compound **7:** Yellow oil; UV (MeOH) λmax 216 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 195.0770 ([M + H]+ (calcd. for C9H11 N2O3, 195.0764); compound **7a**: [α]<sup>20</sup> D 27.7 (c 0.1, MeOH), compound **7b**: [α]<sup>20</sup> D −24.1 (c 0.1, MeOH).

Compound **8:** Yellow oil; UV (MeOH) λmax 276 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 287.0025, 289.0003 ([M + H]+ (calcd. for C10 H12BrN2O3, 287.0026, 289.0005); compound **8a**: [α]<sup>20</sup> D 23.3 (c 0.1, MeOH), compound **8b**: [α]<sup>20</sup> D −20.8 (c 0.1, MeOH).

Compound **9:** Yellow oil; UV (MeOH) λmax 250 nm; 1H and 13C NMR (DMSO-*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 284.9867, 286.9845 ([M + H]+ (calcd. for C10 H10BrN2O3, 284.9869, 286.9849).

Compound **10:** Colorless crystals; UV (MeOH) λmax 240 nm; 1H and 13C NMR (DMSO*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 234.9715, 236.9695 ([M + H]+ (calcd. for C6H8BrN2O3, 234.9713, 236.9692); compound **10a**: [α]<sup>20</sup> D 26.8 (c 0.1, MeOH), compound **10b**: [α]<sup>20</sup> D−32.4 (c 0.1, MeOH).

Compound **11:** Yellow oil; UV (MeOH) λmax 216 nm; 1H and 13C NMR (DMSO*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 235.9920, 237.9899 ([M + H]+ (calcd. for C7H11BrNO3, 235.9920, 237.9899); compound **11a**: [α]<sup>20</sup> D 52.9 (c 0.1, MeOH), compound **11b**: [α]<sup>20</sup> D−46.5 (c 0.1, MeOH).

Compound **12:** Yellow oil; UV (MeOH) λmax 212 nm; 1H and 13C NMR (DMSO*d*6) data, see Tables S3 and S4; HRESIMS *m*/*z* 321.0073, 323.0053 ([M + H]+ (calcd. for C10 H14BrN2O5, 321.0081, 323.0060); compound **12a**: [α]<sup>20</sup> D 20.0 (c 0.1, MeOH), compound **12b**: [α]<sup>20</sup> D −13.3 (c 0.1, MeOH).

Compound **13:** Yellow oil; UV (MeOH) λmax 352 nm; C11 H11 N5O2; 1H NMR (DMSO*d*6) data, *δ*H 12.1 (brs, 1-NH), 8.04 (t, *J* = 4.3, 7-NH), 7.11 (t, *J* = 2.4, H-2), 6.54 (t, *J* = 2.4, H-3), 3.28 (m, H2-8 and H2-9); 13C NMR (DMSO-*d*6) data, *δ*C 164.4, 163.0, 154.9, 129.6, 126.6, 122.6, 121.0, 120.4, 109.6, 39.1, and 31.4. [18]

Compound **14:** Yellow oil; UV (MeOH) λmax 352 nm; C11H11N5O2; 1H NMR (DMSO*d*6) data, *δ*H 11.9 (brs, 1-NH), 7.98 (t, *J* = 4.3, 7-NH), 6.91 (t, *J* = 2.3, H-2), 6.79 (t, *J* = 2.3, H-3), 3.26 (q, *J* = 4.5, H2-8), 2.85 (q, *J* = 4.5, H2-9); 13C NMR (DMSO-*d*6) data, *δ*C 163.9, 161.2, 153.4, 130.5, 126.1, 122.3, 120.4, 118.6, 112.6, 38.3, and 36.6. [16]

Compound **15:** Yellow oil; UV (MeOH) λmax 360 nm; C12H13N5O3; 1H NMR (DMSO*d*6) data, *δ*H 12.0 (brs, 1-NH), 7.76 (dd, *J* = 6.5, 1.7, 7-NH), 7.09 (t, *J* = 2.8, H-2), 6.52 (m, H-3), 5.73 (d, *J* = 6.7, H-9), 3.57, 3.28 (m, H2-8), 3.21 (s, 9-OMe); 13C NMR (DMSO-*d*6) data, *δ*C 164.8, 162.6, 155.8, 130.3, 123.3, 122.9, 127.1, 123.3, 117.5, 110.6, 69.0, and 43.2. [19]

Compound **16:** Yellow oil; UV (MeOH) λmax 214 nm; C12H14BrN5O3; 1H NMR (DMSO*d*6) data, *δ*H 9.78 (brs, 9-NH), 9.44 (brs, 7-NH), 8.10 (brs, 16-NH2), 7.68 (s, H-3), 5.74 (s, H-6), 3.47, 3.36 (m, H2-13), 2.24 (m, H2-11), 1.98 (m, H2-12); 13C NMR (DMSO-*d*6) data, *δ*C 163.6, 163.1, 156.5, 146.0, 118.4, 85.8, 81.4, 63.9, 44.8, 39.5, 19.7 [17].

## *3.4. Computational Section*

Conformational analyses were carried out in the MMFF minimization force field by the Spartan 10 v1.2.4 software package (Microsoft, Redmond, WA, USA). The resulting conformers were optimized using DFT at the B3LYP/6-31+G(d,p) level in the gas phase by the GAUSSIAN 09 C.03 program (Gaussian, Inc. Wallingford, CT, USA). The optimized conformations, whose Boltzmann distributions of Gibbs free energies were more than 1.0 percent, were used for the ECD calculations using the TD-DFT method with the basis set RB3LYP/DGDZVP, or the NMR calculations using the GIAO method at the PCM/b3lyp/6- 311+G(d,p) level.
