**5. Isolation, synthesis, and structural analysis of homoisoflavonoids**

Due to the intrinsic interest in homoisoflavonoids and their biological activities, several works have been discussing different structural aspects of homoisoflavan nucleus‐bearing organic compounds [6, 7]. The structural uniqueness of these compounds and their potent biological activities makes them a target of choice for studies in natural products research on the determination of absolute configurations, organic synthesis, isolation, and structural determination [7].

Homoisoflavonoids classified as protosappanins are commonly associated to the species *C. sappan* and *C. japonica*. These compounds are resulting from the connection of C‐4 and C‐4a atoms forming an eight‐membered ring. There are only eleven protosappanins reported so far [26]. Compounds **31**–**33** did not show significant cytotoxicity against MCF7, A549, LN229 cell lines. In addition, compounds **32** and **33** were also tested against the inflammatory process

**4. Biflavonoids containing homoisoflavonoids subunits in** *Caesalpinia*

Flavonoids can also exist as dimers, named biflavonoids, which represents flavonoids linked by C–C or C–O–C bond in order to form a flavonoid‐flavonoid structure. The connection can occur in several modes in the three rings of the flavan nucleus. The ring A could be linked to the ring A′, indicated as A‐A. This could also occur between the rings A‐C, B‐B, C‐B, among other possibilities that are enlarged by functional groups as OH, MeO, C═O, C═C. The occur‐ rence of common biflavonoids in the genus *Caesalpinia* is known only to some species, such as

Furthermore, certain species from the *Caesalpinia*, mostly *C. sappan*, are associated to the pro‐ duction of biflavonoids containing homoisoflavonoids subunits such as protosappanin D **(37)**, a biflavonoid which exhibit two subunits of **33**, and protosappanin E **(38)**, which display **13** and **33** as subunits. Compounds **37** and **38** were tested against the inflammatory process asso‐ ciated to the inhibition of iNOS and PGE2 production, as well as the suppression of TNF‐α and COX‐2. Washiyama and collaborators suggested that the protosappanin skeleton and the functional group at C7 would be important to the activity of **37** and **38** [27]. The investigation of sappan lignum as a possible XO inhibitor leads to the isolation of diverse compounds including neoprotosappanin **(39)** and protosappanin E‐2 **(40, Figure 5)**. These two compounds presented IC50 of 38.3 and 18.9 μM, respectively, exhibiting a concentration‐dependent behavior. In what extent their mode of inhibition, the biflavonoid **39** was considered a noncompetitive XO inhibi‐ tor while **40**, a competitive inhibitor [2]. The inhibition of XO is associated to improvements in cardiovascular health as well as, the reduction of ROS, and the amelioration of gout cases [36]. The presence of rare caesalpins is correlated to *C. sappan*. A dimer named neosappanone **(41)** was isolated from this species and evaluated against XO. The IC50 of **41** was determined as 29.7 μM and associated to a competitive inhibition of XO. Therefore, these results showed that the traditional use of *C. sappan* for rheumatism and inflammatory diseases could be attributed to its phenolic composition, specifically to dimers of homoisoflavonoids [2].

**5. Isolation, synthesis, and structural analysis of homoisoflavonoids**

Due to the intrinsic interest in homoisoflavonoids and their biological activities, several works have been discussing different structural aspects of homoisoflavan nucleus‐bearing

exhibiting weak to moderate activity [27].

106 Flavonoids - From Biosynthesis to Human Health

*C. ferrea* [33], *C. pyramidalis* [34], *C. pluviosa* [35].

**spp.**

The isolation of homoisoflavonoids involves different chromatographic techniques. Homo‐ isoflavonoids are generally separated after treatment of the organic extract (MeOH, CHCl<sup>3</sup> ) with several chromatographic phases. The use of column chromatography steps (using silica gel and/or Sephadex LH‐20), preparative thin layer chromatography, as well as high per‐ formance liquid chromatography (HPLC) methods (semi‐preparative and preparative) have been used to purification [7]. In addition, there are other methods used to the isolation of flavonoids, such as counter current chromatography [37] can be adopted for the isolation of homoisoflavonoids and flavonoids.

Besides the isolation of naturally occurring homoisoflavonoids from the species *C. pulcherrima*, a synthetic approach of the isolated homoisoflavonoids **1**, **3**, **4**, **5**, **6**, **7**, **9**, and **10** employed the piperidine catalyzed condensation as key steps in this synthesis of these structures (**Figure 6**). This procedure afforded products reaching around 60% yielding following conditions exhib‐ ited in **Figure 3** [19].

The structures of homoisoflavonoids have been unambiguously established by analysis of spectroscopic NMR data supported by analysis of UV and MS spectra. These analyses con‐ firm the presence of the 15‐carbon backbone related to classic flavonoids, and the 16‐carbon skeleton with two phenyl rings (A and C) and one heterocyclic ring (B) separated by an addi‐ tional carbon, forming the homoisoflavonoids skeleton [24, 29, 31].

Analysis of the <sup>1</sup> H and <sup>13</sup>C NMR spectra indicates the presence of carbonyl groups at *δ*C 170.7– 220.0 as well as those assigned to carbons/hydrogens of aromatic ring at *δ*C 100.0–170.0/*δ*<sup>H</sup> 6.00–8.50 and hydroxyl derivate group as characteristic signs. The homoisoflavonoids, when existing as dimers, exhibited their <sup>13</sup>C and 1H NMR spectra typically duplicate and super‐ posed when presenting the same subunits.

The extra carbon existing in homoisoflavonoids compared to ordinary flavonoids can be ali‐ phatic displaying <sup>13</sup>C and 1H NMR signs at *δ*C 30.0–35.0/*δ*H 2.60–3.00; or olefinic at *δ*C 100.0–140.0/*δ*<sup>H</sup> 5.30–6.00, respectively. Correlations in the HMBC, HSQC and COSY spectrum resolve all ambi‐ guities to the structure of these compounds.

The compounds **17**, **19**, **29**, **37**, **38** are homoisoflavonoids derived from the auto‐oxidation of precursors or present differentiated biosynthesis. These compounds present uncommon chemical structures with <sup>1</sup> H, and <sup>13</sup>C NMR spectra relativity complex, in some cases, exhibit‐ ing signs that indicate the presence of lactone and others characteristic group.

Homoisoflavonoids present signals of absorptions in the UV spectrum at *λ*max 222–230, 270–280, and 300–310 nm, depending on the presence of conjugated double bonds on their structures. The MS spectrum present [M+H]+ ions between *m*/*z* 250–350 to homoisoflavonoids and [M+H]+ ions between *m*/*z* 500–600 to dimers of homoisofavonoids with some minor compounds with *m*/*z* > 600 [24, 29, 31].

The absolute stereochemistry can be established by circular dichroism analysis by compari‐ son to models with known stereochemistry. The compound **28**, isolated from *C. sappan*, had its absolute stereochemistry determined by this method compared with data from 3‐deoxysap‐ panol. The result suggested that **28** has the absolute stereochemistry at the C‐3 and C‐4 posi‐ tions to be (3*R*,4*S*) [24].
