This study examines cortical organoids generated from a panel of isogenic trisomic and disomic iPSC lines (subclones) as a model of early fetal brain development in Down syndrome (DS). An initial experiment comparing organoids from one trisomic and one disomic line showed many genome-wide transcriptomic differences and modest differences in cell-type proportions, suggesting there may be a neurodevelopmental phenotype that is due to trisomy of chr21. To better control for multiple sources of variation, we undertook a highly robust study of ∼1,200 organoids using an expanded panel of six all-isogenic lines, three disomic, and three trisomic. The power of this experimental design was indicated by strong detection of the ∼1.5-fold difference in chr21 genes. However, the numerous expression differences in non-chr21 genes seen in the smaller experiment fell away, and the differences in cell-type representation between lines did not correlate with trisomy 21. Results suggest that the initial smaller experiment picked up differences between small organoid samples and individual isogenic lines, which “averaged out” in the larger panel of isogenic lines. Our results indicate that even when organoid and batch variability are better controlled for, variation between isogenic cell lines (even subclones) may obscure, or be conflated with, subtle neurodevelopmental phenotypes that may be present in ∼2nd trimester DS brain development. Interestingly, despite this variability between organoid batches and lines, and the “fetal stage” of these organoids, an increase in secreted Aβ40 peptide levels—an Alzheimer-related cellular phenotype—was more strongly associated with trisomy 21 status than were neurodevelopmental shifts in cell-type composition. Cognitive disability is a universal feature of Down Syndrome (DS), and while the genetic basis of DS is clear—trisomy 21—how an extra copy of this tiny chromosome carrying ∼250 coding genes causes this phenotype is not well understood. Nor is it clear which neural cell-types, tissue and brain regions are impacted, or when impacts occur. Many studies have raised important hypotheses for specific cell phenotypes and mechanisms, but these various findings have not been consistently supported, and in some cases are conflicting. For example, several studies have suggested that interneuron number may be decreased in DS patients and human cell models , in contIn recent years, with the advent of high-throughput sequencing approaches, studies have begun to examine differences in the transcriptomes of DS versus euploid samples, but at this early stage there are few consistent conclusions. Studies almost invariably agree that many chr21 genes are upregulated in DS tissues and cells, although the number and identity of these genes varies between studies. For example, one meta-analysis of 45 transcriptome studies found 77 chr21 genes to be consistently upregulated in DS samples  and a moMost recently, several studies have reported that trisomy 21 causes broad transcriptome-wide changes, with some studies reporting global genomic dysregulation  or the pDYRK1A, RCAN1, OLIG2, and OLIG1, DSCAM, SOD1, PCNT and several IFN-receptor genes) are hypothesized to be central to DS, although consensus has not been reached. Modestly smaller fetal brain sizes have been reported in DS fetuses; however, DS infants have developmental milestones closer to normal at birth and adults often score as more severely impacted than children. Hence, it is important to better understand when in human pre-natal and/or post-natal periods the cognitive deficits arise. This is key to therapeutic strategies, and has been studied primarily in DS mouse models . Some individuals with DS develop amyloid plaques as early as adolescence, and ∼80% show clinical dementia by ∼60–65 years , we identified many subclones that were not transgenic for XIST (but had the tet-puromycin selection gene). Some subclones were shown to be euploid by spontaneous loss of one chromosome 21, with chr21 transcriptome levels equivalent to non-isogenic normal control cells (APP) before each series of differentiations, and trisomy 21 status confirmed by RNA sequencing transcriptomics.The isogenic cell lines used here were generated and characterized as described in our prior study , and expol cells . Severalol cells . iPSCs wAs described in the supplement, we first briefly compared three organoid generation protocols  legend, Forebrain organoids were fixed for 30 min in PFA at room temperature, washed three times with PBS, and cryopreserved in 30% sucrose/PBS at 4°C overnight. Fixed organoids were embedded in O.C.T. compound , frozen in an isopropanol/dry ice slurry, and sectioned at 14 μm on a cryotome. Sections were attached to Superfrost Plus slides  and stored at −20°C until staining. Prior to staining, sections were rehydrated in PBS for 5 min, and detergent extracted in 0.5% Triton X-100  for 3 min.Immunofluorescence was performed as previously described . FixatioWhole organoids were washed once with 1X PBS and placed into 2 ml microcentrifuge tubes containing one 5 mm steel bead  and 1 ml of Trizol reagent . These samples were homogenized using the TissueLyser II instrument  on the P1 setting. Beads were then removed using a magnet and samples were either stored at −80°C or RNA extraction, DNAse treatment, and RNA cleanup was performed immediately.RQN > 7.5 and strand-specific sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina in conjunction with the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Multiplex Oligos for Illumina .RNA was extracted using TRIzol reagent  according to manufacturer’s instructions. RNA samples were cleared of contaminating genomic DNA by DNAse I  treatment for 1 h at 37°C. RNA cleanup and DNAse I removal was performed using RNeasy MinElute columns  according to manufacturer’s instructions. Clean RNA was assessed for quality on an Advanced Analytical Fragment Analyzer. All samples had an Sequencing was performed by the UMass Chan Medical School Deep Sequencing Core Facility on the Illumina HiSeq 4000 platform to a depth of ∼8 million reads/sample (2 × 50bp paired-end) in the case of the large organoid experiment or on the NextSeq 500 instrument to a depth of ∼30 million reads/sample (2 × 38bp paired-end) in the case of the pilot experiment.Reads were aligned to the GRCh37/h19 human genome build using hisat2  (v2.0.5)p-values.  Therefore, to better isolate the evidence for differential expression of genes on or off chr21—a distinction that is a priori relevant for DS studies—FDRs are computed separately for chr21 and for non-chr21 genes, denoted by FDRchr21 and FDRnon21, respectively. Since many studies do not calculate FDR for chr21 and non-chr21 separately, we also report the usual genome-wide FDR in supplemental tables and illustrate the p-value cut-off for FDR < 0.1, along with FDRchr21 < 0.1 and FDRnon21 < 0.1, on the volcano plots in p-value cut-offs are the largest p-values for which the corresponding FDR < 0.1; when none of the N off-chr21 genes satisfied FDRnon21 < 0.1 the p-value cut-off was indicated as 0.1/N, the Bonferroni cut-off—this is more stringent than the FDR cut-off in general, but if a hypothetical single gene were to satisfy the FDR cut-off it would also satisfy the Bonferroni cut-off).The false-discovery rate (FDR)  was usedhttps://data.mendeley.com/datasets/3wrtkk4w5v/2. The Birey dataset uses the Pasca protocol, which corresponds more closely to the protocol used here than the modified Lancaster protocol used for the Quadrato dataset, but this had fewer than 10 cells assigned to several of the reference cell-types. Because of this, the average of the Birey and Quadrato basis vectors was used for deconvolution in the main results reported here. Marker genes for each of the 24 clusters from the Tanaka study were downloaded from https://cells.ucsc.edu/?ds=organoidatlas and ranked by p-value with ties broken by fold-change. For cell-types corresponding to single clusters, the top 30 marker genes were used for the basis vectors. For cell-types comprising more than one cluster, the top five marker genes from each cluster were selected and the remainder of the 30 genes were selected based on rank in combined marker lists for these clusters. Ribosomal and mitochondrial genes (bsecsc function getMITRIB) (Cell-type deconvolution was performed using the DWLS-WLS (dampened weighted least-squares) method in the R package DLWS  based ontMITRIB)  were excFor tests of differential expression with cell-type representation adjustment, the estimated proportion of cortical neurons in the summed replicates was included as an additive covariate in the edgeR statistical model. .t-tests were used—these do not assume equal variance between groups so can better accommodate the reduction in variance that may happen for proportions near zero. In the large organoid study, estimated proportions were based on the summed replicates for each line, for a 3 vs. 3 comparison. Bonferroni correction was used to control for testing for differences in multiple cell-types.For tests of differences in cell-type proportions associated with trisomy 21, Welch’s The R package ggplot2  was used48-h old media was removed from tissue culture wells containing individual organoids. Media was immediately placed on ice and centrifuged at 2,000 rcf for 5 min to remove cell debris. Media supernatant was stored at −80°C. ELISA was performed using the ultrasensitive Amyloid beta ELISA kit from Invitrogen   per manuThe R package drm  was usedP-values and confidence intervals were computed with the contest function in the R package lmerTest (p = 0.71), and similar results were obtained by averaging un-normalized log2 Aβ concentrations for each cell-line and performing a t-test on these 3 vs. 3 averages. This use of RNA levels as a proxy for organoid size/cell-count improves reliability of the comparisons in what are nonetheless approximate Aβ levels in this analysis.Differences in Aβ concentration will be impacted by organoid cell-counts, which we could not directly measure. So, we used RNA concentration as a proxy for cell-count and included it as an additive covariate in a linear mixed-effect model along with fixed effects for trisomy state and batch and random effects for cell line and its interaction with batch [lmer function in the R package lme4 ]. P-valulmerTest  using SaThe results presented detail the progression of experimental design improvements based on results of our initial observations. The last and largest experiment was formulated from lessons learned from our initial cerebral organoid studies, which highlighted the need to address several sources of variation that are often present in iPSC modeling, but not due to trisomy 21. DS studies have reported numerous phenotypic and transcriptional differences attributed to trisomy in human or mouse neural tissues and cells; however, it is often difficult to know whether other potential differences between samples have been ruled out. In our initial experiments, comparing individual isogenic organoids from one trisomic and one disomic line, we found intriguing differences. Given this, we then sought to determine whether these differences might be accounted for by variation (unrelated to trisomy 21) between organoids, experiments, or cell lines—even isogenic cell lines. Realizing the need to expand the experimental design led us to focus on quantitative transcriptome analyses comparing hundreds of organoids from experimental repetitions, and, importantly, generated from additional all-isogenic iPSC lines. While the need for large numbers of organoids to control for variation between organoids was not unanticipated, our results indicate that the number of isogenic iPSC lines needed to confidently investigate a potentially subtle neurodevelopmental phenotype was more than initially expected.Several 3D cell culture models of cerebral development have recently been developed, each with its own set of advantages and drawbacks. We first created organoids using three different protocols  to identWe had initially planned to examine differences between organoids using cytological staining for specific cell-type markers. For example, prolonged culture with this protocol generates significant numbers of GFAP-expressing cells, which include astrocytes  . The figOur first pilot RNA sequencing experiment used bulk RNA-seq of 10 organoids aged for 160 days, five from a trisomic  line and five from an isogenic euploid control line. Isogenic lines have the same genetic background, and comparison of subclones derived from the same iPS parental line avoids differences in reprograming or somatic cell of origin. The overall strategy was to generate bulk sequence data and use published gene sets and expression profiles for different cell-types to deconvolve the cell-type representation in each sample. In this first experiment we chose to evaluate the variation between individual organoids by sequencing the 10 organoids individually.FDR < 0.1 . As explained in the methods, to avoid diluting the signal for differential expression among the small proportion of genes on chr21, we also computed FDRs separately for chr21 genes (designated FDRchr21) and non-chr21 genes (FDRnon21). Here because there were so many DEGs off of chr21 this had only a modest effect, with 111 chr21 genes having FDRchr21 < 0.1 and 5,626 non-chr21 genes having FDRnon21 < 0.1. These 111 chr21 genes included 10 with higher expression in disomic than trisomic samples, which may reflect differences between particular cell-lines or organoid-batches. The DEGs include genes with various non-coding biotypes and genes with lowish average expression, which contribute to the high number of non-chr21 DEGs, but 78%  were for protein-coding genes, 87%  for genes with average CPM > 1, and 75%  for both. As discussed in the introduction, differences of several hundred up to a few thousand non-chromosome 21 genes are a common finding in published studies of DS.We generated RNA-seq data for each of the 10 organoids to a depth of ∼30 million reads and then compared RNA-seq data for the five trisomic and five disomic organoids. This data showed a clear difference in overall expression of genes from chr21 between individual organoids from the trisomic versus the disomic iPSC lines . In testIn p < 0.05 by Welch’s t-test before multiple hypotheses correction) for three cell-types—glial progenitor cells, cortical neurons, and interneuron—but after Bonferroni-correction these were no longer statistically significant, with p ≥ 0.23 for all cell-types   . HoweverEven if consistent significant differences had been seen between organoids generated from these two cell isogenic cell lines, a key question would remain as to whether that difference is due to the presence of trisomy 21, or to other sources of variation between samples, including between isogenic cell lines cultured separately . Since hThe above findings provide suggestive differences in organoid development from the two cell lines, although these were only nominally significant. A more powerful experimental design could affirm or discount that these differences are consistently correlated with trisomy 21. The above results indicated that many more organoids should be examined for each line, but we also recognized that differences observed between any two particular lines could reflect differences unrelated to trisomy 21. Thus, the experimental design was greatly expanded to include many more organoids, more isogenic lines, and more experimental repetitions , in ordeInitial sequencing analysis confirmed the expected ∼1.5 fold higher overall chromosome 21 expression in all trisomic versus all disomic lines .We next used the bulk transcriptome data to estimate the cell-type composition, as in the pilot study. The most highly represented cell-types were cortical neurons and interneurons, which form neighboring clusters in the UMAP projection in the study from which these cell-types were defined . There wp-values > 0.2 before multiple hypothesis correction and all Bonferroni-corrected p-values equal to 1 . Even with several hundred organoids compared in four replicate experiments, the statistical power is limited by the number of isogenic trisomic and disomic lines. Multiple organoids from the same “batch” or replicate batches (experiments) from the same cell line are not independent samples for comparing the effects of trisomy 21, because results will be impacted by the particular size propensity of particular cell lines (or batches) compared.Irrespective of the cell-type composition, we also considered whether differences in organoid size may be correlated with trisomy 21. The presence of the extra chromosome may confer increased cell stress  or cell We emphasize that these findings do not discount that there could be a mild growth disadvantage conferred by trisomy 21, even potentially in organoids, but results here highlight that the inter-cell-line (and inter-batch) variability can be a complicating factor in such studies. In fact, we found decreased proliferation of undifferentiated trisomic iPS cells in a prior study that directly compared cultures of the same cell line with and without induced silencing of one chr21 . Comparip-values that reflect real biological differences between lines or samples, but, importantly, these are not necessarily due to trisomy 21. This is a form of pseudoreplication  of all analyzed chr21 genes. Most chr21 DEGs were at or near the theoretical 1.5-fold higher expression expected in trisomic cells . Neither GFAP nor the DLX genes noted in the smaller pilot study were near the cut-off for significance in the expanded study  of genes expressed at >1 CPM differentially expressed at ic cells ; this ised study .per se. Given the paucity of non-chr21 DEGs in this expanded study, we also considered that inconsistent cell-type representations between the various cell lines/samples will increase expression variability that could weaken detection of gene expression changes between trisomy and disomy groups. To address this, we tested differential expression using a model that accounts for the estimated proportion of cortical neurons in each sample, as this was the cell-type with highest variability between samples (based on variance and IQR). As seen in FDRchr21 < 0.1 from 139 to 143, and genes off of chr21 with FDRnon21 < 0.1 remaining at zero (and just one at FDR < 0.1). Results from tests of differential expression without and with adjustment for cortical neuron proportions are provided in This indicates that the extensive genome-wide differences found in the pilot study  reflect The strongest conclusion from these results is that the presence of widespread differences in expression in the one-line vs. one-line comparison is not validated with a stronger experimental design. Other biological variation, such as in cell-type representation, may be associated with hundreds or more non-chr21 DEGs, not necessarily a direct or indirect effect of trisomy 21. While this overall finding is clear, whether any individual gene is identified as a DEG can be modestly impacted by even small differences in estimated cell-type proportions and specific cut-offs. Thus, changes in the deconvolution methodology, filtering of lowly expressed genes and other computational details can affect whether a few off-chr21 genes are identified as significant, as in our earlier analysis of this data . Hence, Aβ to contrast detection of this neurodegenerative pathology to the neurodevelopmental results. We isolated media from each organoid pool and analyzed whether an increase in secreted Aβ would be detected in these fetal stage organoids, using ELISA for Aβ40 (see Methods). p = 0.068; see Methods) than differences related to neurodevelopment .While an in-depth study of AD-related cell phenotypes is the subject of a separate study, we include here a limited analysis of in utero imaging in large numbers of developing fetuses.This study began with the expectation that isogenic comparisons of trisomic and disomic cortical organoids, as models of early fetal neurodevelopment, would reveal differences in cell-types and pathways caused by trisomy 21. Understanding how and when brain development and/or function is impacted in DS is critical to understanding the biology of trisomy 21 and to assess therapeutic prospects to mitigate cognitive or neurological deficits in DS . Given tNumerous studies report a variety of differences comparing trisomic and euploid samples, including in various mouse models, different human DS tissues, and various cell-based models. Inconsistencies in findings between studies may be due to differences in the systems examined, or in some cases limitations in sample size, etc. Human iPSC studies have the potential to provide more controlled comparisons between trisomic and euploid cells/organoids in comparable developmental/functional states. However, iPSC cultures are prone to environmental changes that can affect experimental results  and orgaFrom the start we used a totally isogenic system, using isolated lines (subclones) derived from a single reprograming event, avoiding the differences between isogenic lines from distinct reprograming events or cell of origin effects, which can manifest as differences in neural differentiation potential . BecauseFDRchr21 < 0.1). This was more than the 58% of chr21 genes satisfying the same criteria in the smaller (pilot) experiment, and more chr21 DEGs than reported in many studies of DS tissues or cells, which, paradoxically—and as in our pilot study—report many more off-chr21 DEGs than found in the expanded experiment here  for each organoid experiment  repository, accession number JC and JL conceived and designed the study. JC performed the experiments. JC, OK, and JL analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.


Medicinal plants have been used by humans since ancient times for the treatment of various diseases and currently represent the main source of a variety of phytocompounds, such as triterpenes. Pentacyclic triterpenes have been subjected to numerous studies that have revealed various biological activities, such as anticancer, antidiabetic, anti-inflammatory, antimicrobial, and hepatoprotective effects, which can be employed in therapy. However, due to their high lipophilicity, which is considered to exert a significant influence on their bioavailability, their current use is limited. A frequent approach employed to overcome this obstacle is the chemical derivatization of the core structure with different types of moieties including heterocycles, which are considered key elements in medicinal chemistry. The present review aims to summarize the literature published in the last 10 years regarding the derivatives of pentacyclic triterpenes bearing heterocyclic moieties and focuses on the biologically active derivatives as well as their structure–activity relationships. Predominantly, the targeted positions for the derivatization of the triterpene skeleton are C-3 (hydroxyl/oxo group), C-28 (hydroxyl/carboxyl group), and C-30  or the extension of the main scaffold by fusing various heterocycles with the A-ring of the phytocompound. In addition, numerous derivatives also contain linker moieties that connect the triterpenic scaffold with heterocycles; one such linker, the triazole moiety, stands out as a key pharmacophore for its biological effect. All these studies support the hypothesis that triterpenoid conjugates with heterocyclic moieties may represent promising candidates for future clinical trials. Since ancient times, humans have used herbs to cure various diseases, and even though they were initially instinctively chosen, people began to know precisely which plants to use for a particular disease due to the experience gained over time . NowadayPentacyclic triterpenes are a well-represented class of phytocompounds resulting from plants’ secondary metabolism, mainly through squalene cyclization ; they cahttp://www.ncbi.nlm.nih.gov/pubmed, accessed on 13 May 2022) and Web of Science . The databases were searched using combinations of the key terms “pentacyclic triterpenes” and “heterocycle”, covering the last 10 years. The inclusion criteria for the current manuscript included: (1) original articles; (2) abstracts or full text available; (3) articles containing data about pentacyclic triterpene heterocyclic derivatives and bioconjugates. The exclusion criteria included: (1) duplicates; (2) articles in languages other than English; (3) review-type papers; (4) irrelevant articles for the topic; (5) articles that discussed only the synthesis of the derivatives without their biological assessment; (6) articles that discussed ring-modified derivatives. Regarding the studies that focused on obtaining not only heterocyclic derivatives but also aryl-derivatives, the review also presents these aryl-derivatives due to the fact that they exhibited significant biological activities in their respective studies, even if they could not be classified as heterocyclic derivatives or bioconjugates. The database screening resulted in a total of 143 articles; however, after the application of the above-mentioned inclusion and exclusion criteria, only 90 articles were subjected to further analysis.The current work aims to summarize the most recent published studies on the topic of semisynthetic derivatives of pentacyclic triterpenes bearing heterocyclic moieties and focuses on biologically active derivatives and their structure–activity relationships (SAR). The studies cited were collected from two major databases recognized by the scientific community, PubMed (Lupeol (lup-20(29)-en-3β-ol; Khan et al. obtained a series of C19-isopropylene-modified lupeol derivatives and further assessed their influence on in vitro glucose absorption. The results obtained for several heterocyclic derivatives either containing a pyrazoline moiety Lup 1–3;  or obtai50 > 70 µM, a Lupeol–C16-sulfate derivative, as well as with doxorubicin used as a standard drug [A series of lupeol derivatives was synthesized by Castro et al. through C3, C16, or C-19-isopropylene chemical modulation, followed by their cytotoxicity assessment against two prostate cancer cell lines, LNCaP and PC-3. Among the tested derivatives, two compounds bearing a double esterification at the C3- and C-16 hydroxyl groups with benzoyl chloride (Lup 12) or p-bromo-benzoyl chloride (Lup 13), respectively, exerted lower cytotoxicity compared with the lead compound ICard drug . The autard drug ; howeverBetula alba, B. pubescens, B. pendula) [Betulin (lup-20(29)-ene-3β,28-diol, betulinol) is a lupane-type pentacyclic triterpenoid bearing two C3, C28-hydroxyle, and one isopropylene moiety at C19 ; this phpendula) . Betulinpendula) . In ordependula) . Saponins represent a heterogeneous class of triterpenoids or steroid glycosides that are widely distributed in nature and exhibit various biological activities, including not only antiproliferative effects against many types of cancers but also antiviral, antifungal, and anti-inflammatory activities . HoweverTomanek et al. tested the biological benefits of 1,4-quinone-conjugated betulin against seven cancer cell lines. Data showed that the quinone moiety attached to betulin at C-28 through a triazole linker was essential for exhibiting anticancer activity; such compounds exerted the strongest anticancer activity against lung cancer (A549 cell line). SAR analysis revealed that the position of the nitrogen atom plays a significant role in terms of future biological effects; thus, compounds (Bet 2–5) that contain a 5,8-isoquinolinedione group exhibited higher activity than those containing a 5,8-quinolinedione moiety. Furthermore, the particular structure of the 1,4-quinone scaffold strongly influences the potency of the biological activity in the following sequence: 2-methyl-5,8-quinolinedione > 1,4-naphtoquinone > 5,8-isoquinolinedione > 5,8-quinolinedione [The triazole linker was also used by Anh et al., who synthesized a series of eleven compounds through click chemistry by coupling C28-betulin-triazoles with the antiviral 3′-azido-3′deoxythydimine (AZT); the compounds were further tested in vitro against KB  and HepG2 (liver carcinoma) cancer cell lines. The results showed that all compounds exerted at least moderate cytotoxic activity against both cancer cell lines; in particular, the hybrid Bet 6 , containThe assessment of betulin bioconjugates in terms of their effects on the mitochondrial membrane potential and, subsequently, on the oxidative phosphorylation was conducted by Dubinin et al., who synthesized a novel betulin conjugate by attaching the (E)-4-(1h Indol-3-yl-vinyl)pyridine fragment to the C-28 bromoalkyl betulin ester (Bet 7) and evaluated its cytotoxic activity in isolated rat mitochondria. The authors previously demonstrated that betulin can solely modify the mitochondrial membrane; in addition, the newly developed betulin conjugate exhibited a strong cytotoxic effect by selectively accumulating in mitochondria, thus leading to the suppression of respiration and oxidative phosphorylation as well as ROS overexpression, finally resulting in cell death . Leishmania infantum growth; by contrast, the addition of carbamate moieties in the same position provided lower inhibitory effects compared with the parent compound. The most active compound against the promastigote forms of L. infantum was 3-hydroxy-20-lupan-29-oxo-yl-1H-imidazole-1-carboxylate (Bet 8), having its C-20-29 double bond oxidized into an aldehyde and displaying an imidazole scaffold grafted at the C-28 position through an ester bond, which revealed an IC50 value of 50.8 μM; furthermore, when administered in combination with miltefosine, the same betulin derivative exerted a synergistic effect, with the IC50 value decreasing to 6 μM. Most importantly, the compound did not cause any significant macrophage necrosis, thus suggesting little to no toxicity to the host cell [Betulin imidazole derivatives have been tested against leishmaniasis, a highly difficult-to-treat tropical disease; Sousa et al. obtained a series of nine betulin derivatives that were further assessed in terms of pharmacological interaction with miltefosine, an antileishmanial drug currently used in polytherapy. According to SAR analysis, the insertion of imidazole moieties at the C-28 position through an ester bond induced highly promising inhibitory effects against ost cell . 50 values within the micromolar range were calculated; moreover, the selected compounds proved to be harmless against healthy human fibroblasts [The insertion of an isoxazole moiety at C-28 through the oxidation of the hydroxyl group followed by condensation and cycloaddition led to a series of compounds that were further tested against MCF7 breast cancer, U-87 MG multiforme glioblastoma, A549 lung carcinoma, and HepG2 hepatocarcinoma cell lines, as well as in hTERT human fibroblasts. The SAR study proved that the substituents attached to the isoxazole ring significantly influenced the overall cytotoxic effects; as an example, the saponin-like derivatives containing glucose substituents grafted on the isoxazole fragment induced cytotoxic effects against A549 and MCF7 cell lines, but no activity was recorded in the HepG2 cells. The most promising results were reported for derivatives bearing small hydrophilic substituents on the isoxazole ring, such as hydroxymethyl (Bet 9–10), 2-hydroxypropan-2-yl (Bet 11), (2-hydroxyethoxy)methyl (Bet 12), carboxyl (Bet 13), and (acetylthio)methyl (Bet-14), for which ICroblasts . P. falciparum malaria parasite. A series of hybrid derivatives were synthesized and analyzed in vitro against malaria parasites as well as human cytomegalovirus (HCMV). The hybrids were obtained with several esterification reactions, either between individual molecules of betulin and artesunic acid or by additionally using ferrocene moieties as linkers. The most active compound was Bet 15, obtained using C28-betulin esterification with one molecule of artesunic acid, which exhibited a 20-fold activity increase compared to botulin, as indicated by the EC50 value of 0.24 μM. However, compared with the effect of chloroquine against P. falciparum, none of the derivatives presented significantly improved antimalarial activity [Betulin and artesunic acid were chosen for the development of new molecules that could effectively treat the drug-resistant activity . E. coli), and eukaryotic cells (rat thymocytes), as well as on rat liver mitochondria. The study showed that all three compounds inhibited the growth of E. coli without exerting a cytotoxic effect on rat thymocytes. However, the presence of the methyl substituents on the pyridine ring was essential for their activity in mitochondria, with a SAR assessment proving that 30-pyridinium-1-yl-lup-20(29)-ene-3,28-diol bromide, which lacks methyl groups, was not able to induce mitochondrial swelling and aggregation; hence, it did not cause membrane permeabilization. Moreover, Bet 16  and S. aureus (minimum inhibitory concentration of 1 μg/mL) [Similarly, three derivatives were synthesized using pyridinium fragments; the conjugates were obtained by attaching the pyridinium fragment to the C-30 allylic bond of betulin, betulonic acid, and betulin 28-O-acetate, leading to the increased polarity of the molecule and creating new amphiphilic betulin conjugates. The antibacterial and antifungal properties of the three derivatives and their triterpenic analogs were tested against both Gram-positive 1 μg/mL) The previously mentioned C28 modulation by using 1,4-quinone moieties can also be conducted in other positions on the betulin scaffold; a large series of betulin-1,4-quinone hybrids were synthesized by conjugating betulin with various compounds bearing the 1,4-quinone fragment through the use of a linker. Different 1,4-quinone fragments were attached to C-3, C-28, and C-30 positions, respectively, of either betulin or betulin derivatives, such as 3-acetylbetulin, 28-acetylbetulin, 28-propionylbetulin, and 30-hydroxy-3,28-diacetylbetulin. All derivatives were tested against a panel of human cancer cell lines, revealing that, in several compounds, the presence of the betulinyloxy group connected to the 1,4-quinone fragment increases their anticancer activity . Furthermore, compound Bet 28, containing a 30-deoxythymidine-5′-yl moiety fragment at C-28, was the only one with antibacterial activity against the two Gram- bacterial strains, K. pneumoniae and E. coli [The neuroprotective activity of several triterpenoid derivatives containing various sugar moieties was tested in vitro on neuron-like SH-SY5Y neuroblastoma cells; the compounds were prepared by attaching triazole-modified sugar moieties through ester bonds to the C30 position in the molecule of C3, C28-diacetylated betulin. The most efficient neuroprotective compound proved to be one betulin conjugate containing a tetraacetyl-β-glucose moiety Bet 23; ; further E. coli .50 = 0.66 μM) by inducing cycle arrest in the G2/M phase. Furthermore, a structure–activity relationship study on the series of the 19 derivatives suggested that the preferential integration of the triphenylphosphonium moiety into the C28 position might optimize the overall cytotoxic effect [The simultaneous modification of both the C-3 and C-28 positions in the betulin molecule may lead to the synthesis of novel mitochondria-targeted derivatives; such derivatives were obtained by attaching triphenylphosphonium moieties to the C-3 and/or C-28 acylated hydroxylic groups, resulting in nineteen betulin conjugates, which have been tested against four cancer cell lines, including human lung carcinoma (A549), human primary glioblastoma (U87), human cervical carcinoma (HeLa), and human colorectal carcinoma (HCT116), as well as one normal colon epithelial cell line (NCM460). After biological assessment, the most promising compound, Bet 29 , which wc effect . 50 of 0.045 μM. A SAR evaluation revealed that the triphenylphosphonium-substituted compounds induced stronger biological activities than both the parent compound and its acylated derivatives. Furthermore, the length of the alkyl chain linked to the ester bond significantly influenced the cytotoxic effect; as such, the methylene group induced lower biological activity compared with propylene and butylene fragments [The impact of grafting triphenylphosphonium moieties to the betulin scaffold was also investigated by Tsepaeva et al.; in a similar manner to the previous study, the triphenylphosphonium moiety was inserted into both the C-28 and C-3 positions, or into the C-28 position alone, through an acyl linker. The anticancer effects of the resulting compounds were assessed against several cancer cell lines: human breast cancer (MCF-7), prostate adenocarcinoma (PC-3), and vinblastine-resistant human breast cancer (MCF-7/Vinb), as well as on human skin fibroblast (HSF). Although most disubstituted compounds exhibited nanomolar range IC50 values, the most active derivative, Bet 30, possessing only one triphenylphosphonium moiety grafted at C-28, exerted superior cytotoxic activity against all tested cancer cell lines, but particularly against MCF-7/Vinb, with an ICragments . Considering the key role heterocycles play as pharmacophores in medicinal chemistry, Csuk et al. synthesized 24 betulin-derived propargylamines by first subjecting betulin to a series of transformations in order to obtain 28-propargyl betulins, which were then further conjugated with different types of N-bearing moieties through Mannich reactions using alkynes, aldehydes, and amines. The C-3 hydroxyl function was altered in some derivatives; hence, several hybrids were betulone derivatives as a result of oxidation, while others had their hydroxyl group previously acetylated. The isopropenyl moiety of the lupane skeleton remained unaffected for all derivatives. The betulin conjugates were further analyzed in terms of anticancer activity against nine human cancer cell lines, allowing for the formulation of some conclusions regarding their structure–activity relationships. A SAR analysis emphasized the importance of the C-28 alkynic function in the biological effect reported for the synthesized compounds. Furthermore, the introduction of N-heterocyclic substituents and amino moieties on the triterpenic scaffold led to significantly improved cytotoxic effects, while 3-O-acetyl hybrids exhibited higher biological activities compared to their deacetylated parent analogs . In order to evaluate the mechanism of mitochondrial toxicity, a series of dimethylaminopyridine derivatives with triterpenoid scaffolds were synthesized by Bernardo et al. by attaching one, two, or three pyridinium moieties to C-3, C-28, or C-30, respectively, in the molecule of betulin (four compounds) or betulinic acid (one compound); the five derivatives were selected from a larger series of previously synthesized compounds  due to s50 = 1.74µM) while moderately acting against normal cells, thus exhibiting some degree of selectivity. In terms of antimicrobial properties, all three betulin-3-yl glycosaminosides have poor-to-no activity against fungi as well as Gram-positive and Gram-negative bacteria [Another approach for the synthesis of novel betulin derivatives consisted of the introduction of 2-amino-2-deoxy-D-glucopyranosyl and -D-galactopyranosyl moieties, respectively, to the C30 position through a glycosidic bond. The resulting compounds, 28-acetylbetulin-3-yl-2-amino-2-deoxy-D-glucopyranoside and betulin-3-yl-2-amino-2-deoxy-D-gluco- and D-galactopyranoside (Bet 34–36), were further tested against the MCF-7 breast cancer cell line and HDFa human dermal fibroblasts, as well as against human pathogenic bacteria and fungi. The three compounds exhibited highly similar structures, the main differences consisting of the orientation of the hydroxyl groups in the sugar fragment and the presence/absence of C28-acetyl esterification. The experimental data indicated that all three saponins induced cytotoxic activities in both normal and cancer cells; however, betulin-3-yl-D-galactosamine (Bet 36) was the most active compound against MCF-7 cells  cells, which showed the highest sensitivity to the newly synthesized compounds [Numerous derivatives were obtained through modifications of ring A; Grishko et al. synthesized a novel series of A-ring-fused heterocyclic betulone derivatives, which were further tested against five cancer cell lines . According to a SAR study, the C(2)-C(3) fusion with oxazole, isoxazole, triazole, and triazine heterocycles, combined with a 28-hydroxyl moiety, induced far superior cytotoxic activities compared with their 28-acylated azole precursors. Moreover, the oxidation of the 28-hydroxyle moiety in the triazole-fused derivative led to significant suppression of the biological activity, while the presence of a 28-carboxyl fragment combined with a triazine moiety attached to the triterpenoid skeleton highly improved the cytotoxic effect. The study identified N-acetyltriazole betulin as a lead compound (Bet 41) due to its strong antiproliferative effect against all tested cancer cell lines, but particularly against HCT 116  and other inflammation markers. According to a SAR, the conjugates obtained by fusing a heterocyclic group to ring A exerted the highest inhibitory activity, especially when pyridine or pyrazine moieties were used. Moreover, the derivatization of the C-28 hydroxymethyl moiety into an oxime group significantly improved the compound’s biological activity; by contrast, the indole derivatives demonstrated weaker activity compared with the parent compound. The study revealed that compound Bet 42 (pyrazole A-fused betulin derivative), having an oxidized C-28 hydroxyl function, exhibited the highest in vitro inhibitory effect on carrageenan-induced paw inflammation in mice, as indicated by the reduction in nitric oxide production as well as the expression of pro-inflammatory cytokines and prostaglandin synthase-2 (COX-2) .Betulinic acid -en-28-oic acid; 50 = 7.80 nM [Wang et al. synthesized a BA-nucleoside bioconjugate (BA 1) through a click chemistry reaction between the C2-propargyl derivative of BA and 4′-azido-2′-deoxy-2′-fluoro-β-D-arabinocytidine—which exerted potent anti-HIV activity in vitro compared with the parent compounds—with an IC 7.80 nM . Further 7.80 nM . Altoget50 values of 1.62 µM and 1.19 µM, respectively. The SAR evaluation revealed that the introduction of aromatic N-heterocycles (pyrazine and pyridine) is unfavorable for the cytotoxic effect of the resulting derivative. In contrast, the introduction of saturated N-heterocycles (pyrrolidine and piperidine) significantly increases cytotoxicity, particularly against the multidrug-resistant cell line MCF-7/ADR [50 values ranging from 0.5 to 2.4 μM. A SAR evaluation also revealed that, in the series of C3-picolyl-BA derivatives (BA 4–6), the o- and m-substitution of the pyridine heterocycle favors cytotoxic activity, while in the series of C28-picolyl derivatives (BA 7–9), the p- and m-substitutions increase their cytotoxicity as opposed to the o-substitution [Several nitrogen-containing heterocycles were conjugated with BA by reacting the triterpenic acid and C3-amino-BA with various acids bearing pyrrolidine, piperidine, pyrazine, and pyridine moieties; all compounds were tested in terms of cytotoxicity against a panel of eight non-drug-resistant , one multidrug-resistant (MCF-7/ADR), and one normal  cell lines. The tests revealed two amide derivatives, compounds BA 2 and BA 3, each containing one piperidine moiety, which have shown high selective cytotoxicity against the above-mentioned cancer cell lines with average ICCF-7/ADR . Complemtitution . Collect50 = 5 µM) compared with BA, while the tri- and tetra-rhamnose derivatives expressed no sign of cytotoxicity in tested cells [50 > 50 µM), a reaction intermediate, the C28-benzyl ester of the BA-diosgenin bioconjugate (BA 12), showed promising effects against the CEM cell line, with an IC50 = 6.5 µM [50 of 4.25 µM after photostimulation in the presence of glutathione [The general approach to optimize BA bioavailability through chemical derivatization is to graft hydrophilic groups to the core structure in order to increase its hydrophilicity. Thereby, Sylla et al. obtained a series of C3-O-rhamnose-BA conjugates and tested their in vitro effects against DLD-1  and WS1 (skin fibroblast) cell lines. Among the evaluated bioconjugates, compound BA 10, 3-O-α-L-rhamnopyranosyl-(1->4)-α-L-rhamnopyranosyl-BA, exerted selective and superior cytotoxicity against colorectal cancer cells  was found to be the most active in vitro and was further associated with miltefosine, an antileishmanial drug, in order to identify a potential synergistic activity; unfortunately, the compound induced a slight decrease in the ICne alone . When anne alone . Among tne alone , who tes50 = 0.89–7.44 µM), as well as lower cytotoxicity against the tested non-cancer cells (IC50 = 17.76–27.21 µM) [50 values within the micromolar range , HepG-2 (hepatocarcinoma), BGC-823 (gastric cancer), and SY-SY5Y (neuroblastoma) cell lines. The results revealed the following aspects regarding the SAR in the series: (1) the esterification of C28 with benzyl alcohol is favorable only when the C3 position simultaneously contains a piperazine heterocycle (BA 22)—otherwise, it leads to inactive derivatives; (2) the oxidation of C3 into a keto group, combined with the introduction of a cyclohexyl-amino-ethyl, cyclopentyl-amino-ethyl, or 1-ethyl-piperidine moiety (BA 23–25), at the C28 position is beneficial for a cytotoxic effect against HeLa and SK-SY5Y cell lines . A BA-C27.21 µM) . Therefoar range  and reve50 values ranging between 0.81 and 2.62 µM [In a similar manner to the previously presented betulin, the triazole moiety was exploited for the derivatization of BA; a series of N1-substituted-1,2,3-triazole compounds were used as esterification partners for the C28-carboxyle group, resulting in derivatives with potential anticancer effects. In vitro tests against 4T1 (breast cancer) and MIA-PaCa-2 (pancreatic cancer) cell lines allowed for a SAR analysis, which revealed that the free C3-OH is essential for the cytotoxic activity since its oxidation into the keto group or esterification with succinic acid significantly reduced such effects. Furthermore, it was also noticed that some derivatives containing either pyrazinyl or indolyl moieties fused at the C2-C3 positions did not produce a significant effect on tested cells. Collectively, these findings enabled the selection of four lead molecules, each bearing a free C3-OH group and an N1-substituted-1,2,3-triazole moiety at the C28 position (BA 31–34), all exhibiting IC 2.62 µM . In the  2.62 µM , thus su50 values below 3.0 µM against MCF-7 (breast cancer) and TET21N (neuroblastoma) cell lines. The acetylation of C3 alone resulted in one BA derivative with low in vitro cytotoxicity, thus highlighting the key role played by the cationic group in exercising the cytotoxic effect [50 = 2.40 µM) compared with the parent BA, the authors reported the preparation of a semisynthetic compound with low selectivity toward cancer cells [50 ranging from 3 to 7.5 µM. The active conjugate differed from the other compounds by being substituted with two hexyl fragments of the safirinium P moiety, which is linked through a piperazinyl ring at the C28 in BA scaffold, a substitution that could favor the transmembrane passage compared with substitution with ethyl or dodecyl moieties. Interestingly, while it is well known that BA possesses mitochondria-targeted effects and substitution with cationic moieties usually boosts such effects, the study revealed that the safirinium core could not penetrate the cell membrane; therefore, its BA-conjugate accumulated inside the endoplasmic reticulum, which seems to mediate its cytotoxicity [The synthesis of new derivatives using butanoic or triethylene glycol as spacers to attach a cationic triphenylphosphonium group at the C28 position of BA was conducted by Nedopekina et al., who introduced the acetylation of the C3 position in some derivatives, thus achieving cytotoxic derivatives (BA 36–40) with ICc effect . Likewisc effect ,72 of BAc effect . The trier cells . The cattoxicity . Collect50 = 5.9–8.5 µM) against either KB or HepG2 cells compared with the parent compounds. Of note, two of these compounds were also simultaneously subjected to C3 structural modifications: esterification with succinic acid (BA 48) and oxidation into the keto group (BA 50), thus suggesting that these alterations could add to overall cytotoxicity against tested cells [Plasmodium falciparum-infected erythrocytes and human cytomegalovirus (HCMV) hosted inside human fibroblasts. The study showed that one derivative obtained through the esterification of the C3 position of BA with artesunic acid (BA 51) exhibits a potent antimalarial and antiviral activity. Although the derivative showed stronger antimalarial effects compared with BA, the compound was less potent compared with artesunic acid and chloroquine phosphate, the latter being used as a standard drug for the antimalarial effect. Moreover, the antiviral effect of the same derivative was superior to both BA and the standard antiviral drug ganciclovir [One of the most frequent approaches in drug design consists of the synthesis of bioconjugates containing two bioactive components in order to reach an overall increased therapeutic effect, based on the combination of two individual mechanisms of action. In this line of investigation, Bori et al. synthesized an ester bioconjugate (BA 46) of bevirimate with azidothymidine, a nucleoside reverse transcriptase inhibitor that is linked through a 1,2,3-triazole moiety in the C28 carboxyl group of bevirimate; the bioconjugate revealed superior anti-HIV properties compared with the semisynthetic triterpenoid . Within ed cells . Furthered cells . This hyciclovir .50 = 1.30–2.24 µM) compared with BA, thus suggesting that C3 acetylation combined with C28-grafted linear alkyl amide could enhance the transmembrane passage and, consequently, the activity of the bioconjugate [Previous studies have found the combination of BA and cisplatin to exert a synergic cytotoxic effect against several cancer cell lines ,80. In oonjugate . Continuonjugate . These i50 = 5.20 µM) [50 = 4.70 µM and OA-CD 2: IC50 = 6.50 µM) differ only by the presence of a methyl or hydroxyl substituent on the triterpenic scaffold, showed the most promising antiviral effects without associated toxicity against host cells, thus suggesting the occurrence of high antiviral selectivity that should be further investigated [Given the assumption that the poor BA oral bioavailability is caused by its low water solubility, BA was conjugated with macromolecular compounds, such as cyclodextrins (CD), in order to achieve higher hydrophilicity. Although cyclodextrins are frequently used to incorporate lipophilic molecules, such as triterpenic acids, inside their inner cavity, the outer surface could also be used to obtain numerous derivatives with enhanced biological activity . One of 5.20 µM) . Continu5.20 µM) . The aspstigated .Although the literature on the topic highlights many BA derivatives with superior biological activity, studies that have obtained derivatives with diminished activity compared with either BA or standard cytotoxic drugs should also be brought to attention in order to limit the waste of resources for fellow researchers. For example, Khlebnikova et al. obtained C28-modified F-containing-2-acylcycloalkane-1,3-diones-BA conjugates with lower cytotoxicity against MCF-7 (breast cancer) and U-87 MG (glioma) cells compared with doxorubicin . Similar50 = 3.3 µM), K562 (IC50 = 3.6 µM), and K562-TAX (IC50 =3.9 µM) [Staphyloccocus aureus compared with the parent compound, thus indicating that aromatic or liphatic N-containing heterocycles could be used for the synthesis of antimicrobial BA derivatives [Given the reported key pharmacophore role played by the triazole moiety, it was employed by Sidova et al. in the synthesis of BA-4-substituted-1,2,3-triazole derivatives via Cu-catalyzed Huisgen 1,3-cycloaddition to the C30 allylic group using C3 acetylated BA-derivatives as starting compounds; when tested in vitro against a panel of eight cancer and two normal cell lines, compound BA 53, containing an acetylated C3 and a 4-(2-formylphenyl)-1,2,3-triazole in the allylic position, showed significant cytotoxicity as well as selectivity against several types of leukemia cells such as CCRF-CEM (IC=3.9 µM) . The insivatives .Another previously reported promising approach was the insertion of sugar moieties on triterpenoid scaffolds; therefore, among a series of C30-N-acetyl-D-galactosamine-triterpenes conjugates, two BA derivatives (BA 56–57) were selected and investigated in terms of cytotoxicity, revealing, however, inferior properties compared with BA and doxorubicin; despite this apparent failure in research, the two compounds showed an improved affinity for the asialoglycoprotein receptor (ASGPR-receptor), expressed mainly in mammalian hepatocytes, which is currently the target for the development of hepatotropic drugs . FurtherIn an effort to identify new pharmacophore groups for the chemical manipulation of BA, a series of biotinylated BA bioconjugates were synthesized through derivatizations in C3, C28, or C30; the bioconjugates revealed the same level of cytotoxicity as the parent compounds against CCRF-CEM (leukemia) and HCT116  cell lines ; howeverBA conjugates can also be obtained by fusing various heterocycles to the C2-C3 bond in ring A of the triterpene scaffold; among such conjugates, Xu et al. identified one derivative containing a pyrazole fused to the A-ring of BA, SH479, which showed remarkable activity regarding osteoclast differentiation compared with BA and, therefore, could be used in the treatment of osteoporosis . In addiBetulonic acid -en-28-oic acid; The introduction of piperidine or pyrrolidine nitroxyl to the C28 position of the BoA molecule through the formation of amidic bonds may result in the occurrence of optimized hepatoprotective properties compared with the parent compound. Sorokina et al. reported the synthesis of a piperidine–nitroxide derivative BoA 1;  and a pyAnother important heterocycle found in BoA derivatives with anti-inflammatory properties is the previously mentioned triazole. Vasilevsky et al. synthesized several BoA derivatives with anti-inflammatory activity, containing a substituted 1,2,3-triazole moiety at the C28 position; the activity has been attributed both to the triterpenoid scaffold and the sidechain containing the triazole moiety. The SAR evaluation revealed that the anti-inflammatory effect increases upon replacing the hexyl substituent of the triazole moiety with an aryl substituent, in particular, an acetylphenyl group, which appears in the structure of the lead compound, BoA 6 . Similar50 = 0.17 μM, an effect that has been associated with its decrease in lipophilicity [50 = 2.4 μM [The triazole heterocycle was also highlighted as a key pharmacophore in the structure of various BoA derivatives with anticancer properties. A series of derivatives of 3-acetylbetulin and betulone containing a 1,2,3-triazole moiety were synthesized through a “click” reaction in the C28-COOH position of triterpenic compounds and assessed in terms of cytotoxicity against three cancer cell lines, C-32 (melanoma), T47D (breast cancer), and SNB-19 (glioblastoma). The study reported that, as a general rule, the substitution of the triazole moiety with carbonyl or acyl substituents such as acetyl induces stronger anticancer activity against the C-32 amelanotic melanoma cell line compared with substitution with aryl substituents. Moreover, a BoA conjugate with 3′-deoxythymidine-5′-yl (BoA 9) proved to be the lead compound against the SNB-19 (glioblastoma) cell line, with an IChilicity . In addi= 2.4 μM . Another= 2.4 μM . Unfortu= 2.4 μM . A series of indole- and pyrazine-fused ring A derivatives of lupane, oleanane, ursane, and dammarane-type triterpenoids were synthesized from their corresponding 3-oxo-derivatives; the C2–3 indole triterpenoid derivatives were obtained by Fischer indolization of 3-oxo phytocompounds with phenylhydrazine, while the C2–3 pyrazine derivatives were obtained by reacting the 3-oxo compounds with ethylenediamine. The newly synthesized hybrids were tested for their inhibitory activity against α-glucosidase, allowing some conclusions to be drawn in terms of SAR; thus, a SAR analysis revealed that the main triterpenic core significantly impacts the inhibitory activity, as indicated by the higher efficacy of lupane, oleanane, and ursane indole derivatives compared with their allobetulin and dammarane indole analogs. In addition, the activity of 2,3-indolotriterpenoids exceeded that of their corresponding 3-oxo-derivatives; particularly, 2-3 indolobetulonic acid (BoA 14) exhibited strong α-glucosidase inhibitory effects compared with BoA, proving to be 221-fold more active than acarbose .Ursolic acid (UA) is an ursane-type triterpenoid found in nature either in its free form or as the aglycone of triterpenoid saponins; it can be extracted from a large a variety of plants, but especially from apple juice; moreover, it was reported to exhibit a wide spectrum of pharmacological activities, including antitumor, antioxidant, anti-inflammatory, cardioprotective, antiviral, etc., effects . Taking 50 values ranged between 9.8 and 16 μM, indicating that the anti-inflammatory effect of each compound depends on the number of sugar units attached to the main skeleton [The rhamnose moiety, previously reported as part of the pharmacophore group due to the cytotoxic activity of other triterpenes, was also used for the derivatization of ursolic acid; the resulting saponins were subjected to in vitro tests against human colorectal adenocarcinoma (DLD-1) cells and healthy human skin fibroblasts, while their anti-inflammatory activity was quantified by assessing the NO production. The experimental data showed that the presence of a single or di-rhamnose unit did not improve the overall cytotoxic activity of the molecule compared with ursolic acid; moreover, the presence of three- and tetra-rhamnose units completely abolished the compounds’ cytotoxicity. In contrast, all derivatives, UA 1–4 , effectiskeleton . 50 = 22.9 µM) and UA 6 (IC50 = 1.55 µM) containing one furoxan ring linked to the triazole moiety. In addition, UA 6, obtained from UA 5 by acetylating the C-3 hydroxyl function, showed higher selectivity (S.I. = 6.72) for MCF-7 cells compared with both ursolic acid and doxorubicin. However, their biological activity was less effective against U-87-MG (multiforme glioblastoma), HepG2 (liver carcinoma), and A549 (lung carcinoma) cells compared with the parent compound. Moreover, the SAR analysis revealed that the introduction of an additional ester spacer between the triazole group and the main skeleton led to a significant decrease in biological activity. Betulonic acid derivatives with furoxan functions linked to the main skeleton through an amino acid linker showed no cytotoxic properties against the tested cancer cell lines. However, 1,2,3-triazole and 1,2,5-oxadiazole betulone hybrids showed selective cytotoxic activity against MCF-7 cell lines compared with the parent compound, demonstrating the importance of the triazole and oxadiazole functions toward the biological activity of the compounds [The relevance of nitrogen-containing heterocycles regarding the cytotoxic effects of semisynthetic triterpenes was confirmed for ursane and lupane derivatives as well. 1,3,4, and 1,2,5-oxadiazole and furoxane moieties were attached to the triterpenoid scaffold through plain or modified triazole linkers, which form esteric bonds with the C3 or C28 carboxyle groups. Some derivatives contained a combination of triazole and oxadiazole functions attached to the ursane and lupane skeletons via a succinate linker. The furoxane derivatives presented an additional ester linkage bond that connected the triazole linker and the furoxane fragment to the C-28 position of the triterpenoid frame. In vitro tests against several cancer cell lines revealed the strong cytotoxic activity in MCF7 cells (breast carcinoma) of compounds UA 5 , MCF-7 (breast cancer), HT-29 , and A2780 (ovarian adenocarcinoma). The results revealed that the parent triterpenoids showed little to no cytotoxicity against any cell line. However, the most effective cytotoxic effects against all cancer cell lines were proven for all 3-O-acetylated piperazinyl derivates amidic derivatives. The BODIPY hybrids exhibited little cytotoxicity; only the 3-O-acetyl-betulinic acid-derived BODIPY FL conjugate of UA (UA 7) exhibited a significant cytotoxic effect against the MCF-7 cell line with an EC hybrids .Lagerstroemia speciosa L. (banaba) with significant pharmacological potential in treating diabetes due to its insulin-like properties, which made it known as phyto-insulin [Maslinic acid (MA) is a pentacyclic triterpene occurring in several vegetables and fruits that are part of the Mediterranean diet, exerting a wide spectrum of pharmacological activities such as antitumor, antidiabetic, cardioprotective, neuroprotective, antiparasitic, and growth-stimulating effects . Corosol-insulin ; in addi-insulin . The mos50 = 499.06 μM) compared with acarbose (IC50 = 606 μM), thus revealing the importance of the free hydroxyl function in the piperazine ring regarding the hypoglycemic activity. This behavior led the authors to the conclusion that the presence of a free hydroxyl or amino group in the piperazine moiety attached to the main skeleton significantly enhances the α-glucosidase inhibition. In a series of 1-deoxynojirimycin derivatives, it was noticed that CA conjugates exerted higher α-glucosidase inhibitory activity compared with MA derivatives, while both were more effective than the parent compounds. Moreover, a SAR assessment revealed that the length of the carbon chain linker plays a crucial role in the hypoglycemic activity of the compounds; hence, a longer hydrophobic carbon chain causes a decrease in the inhibitory activity [One of the biggest challenges in managing type 2 diabetes is to control the amount of carbohydrate intake; inhibitors of α-glucosidase might ameliorate hyperglycemic episodes by inhibiting the conversion of oligosaccharides into monosaccharides, which, in turn, trigger high blood sugar. Unfortunately, conventional α-glucosidase inhibitors fail to provide high patient compliance due to several side effects, thus creating the need to develop more natural alternatives to modulate blood sugar levels . Such naactivity .Oleanolic acid  is an oleanane-type pentacyclic triterpenoid that can be found in nature as a free acid or as the aglycone of triterpenoid saponins . The phyReferring only to the period of time covered by the present review, one must cite the study of Medvedeva et al., who synthesized lupane- and oleanane-type triterpenoids by conjugating azidothymidine fragments to the main skeleton at C-17 and C-20 through 1,2,3- triazole linkers; when tested against several cancer cell lines, including HCT-116 colon, A498 kidney, PC prostate, HL-60 leukemia, RPMI-8226 myeloma, and SR lymphoma cancer cells, the results revealed that most compounds exhibited high cytotoxic activity against all types of cancer cells. Moreover, OA 1 , C-28 (hydroxyl/carboxyl group), and C-30  or the extension of the main scaffold by fusing various heterocycles with the A-ring of the phytocompound. Many such derivatives also contain linker moieties that connect the triterpenic scaffold with their reaction partners; one such linker, triazole, stood out as a key pharmacophore for the overall biological effect.Considering the high number of studies that aim to achieve the feasible synthesis of compounds of natural origin with significantly improved pharmacological activities, one may hope to discover suitable alternatives to current treatments. However, there are still important issues that need to be addressed. Firstly, more elaborate in vivo studies have to be designed in order to assess the safety and efficacy of the newly synthesized conjugates; secondly, the underlying mechanism of action for both the semisynthetic derivatives, as well as their natural parent compounds, has yet to be fully elucidated. Despite these challenges, triterpenoid conjugates may represent promising candidates for future clinical trials.


Commercial multicolour fixed immunophenotyping panels can improve flow cytometric diagnostic immunophenotyping repertoire.This study validated the commercially available, standardised Beckman Coulter lyophilised DURAClone RE panels to discriminate specific haematolymphoid subtypes.n = 11) or normal bone marrow haematogones (n = 9); CLB panels established haematolymphoid subtypes of mature B-cell lymphoproliferative disorders (B-LPD) (n = 20), while PC panels detected plasma cell dyscrasias (PCD) (n = 17). Flow cytometer setup and data interpretation to discriminate normal and aberrant immunophenotypes were per manufacturer’s instructions.We compared the diagnostic capability of the DURAClone acute leukaemia B (ALB), chronic leukaemia B (CLB), and plasma cells (PC) panels to the predicate second-line panels in Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg, South Africa, from April to August 2020. Clinical diagnostic concordance between the in-house second-line immunophenotyping (the predicate method) and DURAClone was established. The ALB panels tested for precursor B-cell acute lymphoblastic leukaemia , mature B-LPD  and PCD.The DURAClone CLB exceeded the predicate second-line performance, offering extended second-line diagnostic discernment of mature B-LPD subtypes and discernment of CD5+ B-LPD from other non-CD5+ (or CD5–) B-LPD; likewise, the PC panels enabled discovery of PCD. While ALB testing offered no additional diagnostic advantage over existing predicate investigation, CD58 did offer additional information to discern haematogones from B-ALL. Multicolour fluorochrome-conjugated antibody panels generally require antibody concentrations and fluorescence intensity optimisation through titration techniques.5 These assays can be technically challenging and error-prone due to the addition of multiple individual liquid reagents requiring manual pipetting,6 thus requiring laboratory technicians skilled in titration technique and flow cytometry,7 including fluorescence-minus-one, colour compensation, and other technical flow cytometry issues.Flow cytometry immunophenotypic analysis is a powerful diagnostic and research tool for investigating haemato-lymphoid malignancies, such as leukaemia, lymphoma, and plasma cell dyscrasias (PCD). Recently, multicolour flow cytometric immunophenotyping has become invaluable for improving detection and detailed immunophenotyping during diagnosis and detection of low frequencies of abnormal or aberrant cell populations during minimal residual disease assessment.13 like the Beckman Coulter (BC) DURAClone inventory, including the identification of normal and abnormal B progenitor cells (RE ALB),14 various chronic B-cell lymphoproliferative disorders including CD5+ chronic lymphocytic leukaemia (RE CLB)15 and normal or abnormal plasma cells (RE PC) panels, offer an attractive alternative to the locally developed, in-house multicolour panels described. Commercial multicolour panels also usually offer the advantage of a standardised instrument and analysis protocols; preset software-guided controls are available to establish optimal standardised instrument settings, verified with abnormal and normal stabilised blood material controls.9 In addition, these commercial panels are typically fixed panels supplied in a stable, dry (lyophilised) format with predetermined antibody combinations in a single tube9; these qualities enhance laboratory efficiency, reduce technical laboratory error and simplify reagent inventory. Some commercial panels also offer some flexibility in marker combinations by providing ‘open’ fluorescence channels15 so that liquid drop-in markers can be added to the ‘fixed’ panels to accommodate unique and specific diagnostic requirements of the investigating laboratory.Commercially available, standardised, lyophilised, fixed and pre-titrated antibody panels,16 multicolour panel system . Although this method provides excellent coverage to discover most acute leukaemias,16 the ClearLLab 10C system lacks additional markers to further identify and discern specific haematolymphoid subtypes, especially within the group of mature B-cell lymphoproliferative disorders and PCD. Specifically, although the BC ClearLLab 10C can easily discern normal B-cell precursors or haematogones from similar immunophenotypes present in minimal residual B-cell acute lymphoblastic leukaemia (B-ALL) disease, the CD58 marker is not included. However, it may be helpful to discriminate disease from normal B-cell precursors during minimal B-ALL disease assessment.2 The ClearLLab 10C system also lacks markers to discern CD5-positive and CD5-negative mature B-cell lymphoproliferative disorders (LPD), using markers such as CD23, CD43, CD79b, CD81, and ROR-1 recommended for classification by Rawstron et al.18 Investigation of PCD is also limited if investigated only with the ClearLLab 10C system which lacks CD138, an absolute requirement for identifying plasma cells together with CD3819. In our laboratory, we have developed several 2–4-colour in-house, second-line immunophenotyping panels to identify the specific haematolymphoid subtypes mentioned, including markers such as CD23 and FMC-7 for mature B-cell LPD and CD138 with CD56 and CD200, to diagnose PCD. These, however, require a separate repertoire of liquid reagents and necessary flow cytometry technical expertise to set up and run the tests. For a busy site, such as our unit, which processes over 400 samples per month, standardised commercially available panels that can supplement second-line immunophenotypic investigation after ClearLLab10C investigation could dramatically increase the repertoire of markers tested and also improve workflow efficiency in our site.Our laboratory previously established the positive impact on quality, laboratory workflow and diagnostic usefulness of the commercially available, fixed and standardised BC ClearLLab 10CExisting predicate second-line investigation utilises locally established, in-house, non-standardised and manually assembled liquid fluorochrome-conjugated antibody panels. The primary aim of this study was to evaluate the fixed and standardised, pre-titrated commercially available DURAClone RE ALB, RE CLB and RE PC panels  as a comprehensive and compact alternative to our existing second-line immunophenotypic investigation of haematological neoplasms. There were two parts to this study. First, we compared the DURAClone CLB and PC tubes to the laboratory’s in-house predicate 2–4-colour method for the following markers: combinations of CD10, CD23, FMC7 and CD22 to discern mature B-cell lymphoproliferative disorders, or CD19, CD38, CD56, CD200 and CD138 to characterise plasma cells. Secondly, we verified manufacturer-described expression for those additional markers that were not included in the older predicate 2–4-colour method, such as verifying the expression of CD43, CD79b, CD81 and ROR-1 in the CLB tube and CD38 and CD138 with CD27, CD81 and CD56 included in the DURAClone PC tube against expected expression in normal leucocyte population counterparts. We also validated the overall diagnostic outcome of the ALB tube; here, we asked whether the CD58 included in the ALB tubes offered any additional diagnostic advantage in identifying a B-ALL that had been identified in the first-line ClearLLab 10C investigation.The University of the Witwatersrand Health Research Ethics Committee approved the study (Ethics Clearance number M1704129). The study’s objective was to validate the commercially available Beckman Coulter DURAClone PC, CLB and PC fixed-tube, pre-titrated panels as an alternative second-line diagnostic workup to our existing in-house second-line immunophenotypic investigation. The interpretation of flow cytometric data and consequent clinical diagnostic outcomes of the existing in-house, second-line, non-standardised panels were compared to the overall clinical diagnostic outcomes of the DURAClone analyses.20 The work was undertaken from April 2020 to August 2020 and conducted in the National Health Laboratory Service flow cytometry laboratory at the Charlotte Maxeke Johannesburg Academic Hospital in Johannesburg, South Africa. This laboratory performs routine immunophenotypic investigation of leukaemias and lymphomas for both children and adults, referred from Charlotte Maxeke Johannesburg Academic Hospital and other hospitals within the University of the Witwatersrand service precinct, as well as from other centres around South Africa. Morphological assessment of peripheral blood, bone marrow and trephine samples (from the same patients whose samples are tested in the flow cytometry unit) is performed in the sister haematology laboratory; auxiliary molecular and cytogenetic investigations are also performed on site. The laboratory participates in the United Kingdom National External Quality Assurance Scheme  Leukaemia Immunophenotyping Part 1 and Part 2 (interpretation) proficiency testing scheme.21This study was a prospective observational cohort study conducted according to the Standards for reporting Diagnostics Accuracy.17 or PCD22 requires second-line testing using markers that are not included in the first-line testing. For this study, in-house second-line testing specifically assessed CD23 and FMC-7 expression on mature B-cells or CD38 and CD138 expression on plasma cells. Specifically, in the context of B-LPD and in comparison to our in-house second-line investigation that included CD23 and FMC-7, could DURAClone CLB testing with ROR-1, CD20, CD43, CD79b, and 81 replace CD23 and FMC-7 in discerning CD5 B-cell chronic lymphocytic leukaemia (CLL) from other CD5 and non-CD5 expressing B-LPD.18Immunophenotypic testing of all samples at the Charlotte Maxeke Johannesburg Academic Hospital flow cytometry unit is two-tiered. Firstly, an initial first-line workup is performed with BC ClearLLab 10-C . The first workup identifies most acute myeloid and lymphoid leukaemia subtypes, distinguishes early and mature T-cell or B-cell lymphoproliferative disorders, and hints at the presence of a PCD (if a population of brightly expressing CD38 cells is noted). A limitation of this ClearLLab 10C system is that further immunophenotypic characterisation of mature B-cell lymphoproliferative disordersn = 13), bone marrow (n = 43), and a pleural fluid sample (n = 1) collected from paediatric and adult patients. For DURAClone ALB testing, 20 bone marrow aspirate samples were tested. Eleven of these patients had a diagnosis of B-ALL (n = 11), while nine had immunophenotypically normal haematogones. One of the nine was a follow-up of a B-ALL with normal haematogones and no evidence of residual disease. Twenty samples diagnosed with a B-cell lymphoproliferative disorder by predicate ClearLLab 10C and in-house second-line were referred for DURAClone CLB testing. The CLB testing set comprised 12 bone marrow aspirate samples, seven peripheral blood samples, and a single pleural fluid sample. Due to the decommissioning of the older FACSCalibur flow cytometer during the period of study (the instrument used to undertake the predicate in-house second-line testing), only 17 samples with a diagnosis of a PCD by ClearLLab 10-C and in-house second-line investigation were tested by DURAClone PC panels.After all routine testing with ClearLLab 10C and in-house second-line testing , samplesTM  and the FACSCalibur flow cytometer  were used during this study. The NaviosTM flow cytometer was used to analyse the predicate ClearLLab 10C as well as the test DURAClone panels. Internal quality control on the NaviosTM included daily assessment of background contamination, cellular events carryover between tubes, and fluorospheres acquisition to verify the flow cytometer optical alignment and fluidics . In addition, ClearLLab™ Normal and Abnormal process control cells  were used to verify sample processing, acquisition, and analysis. ClearLLab 10C panel acquisition setup was achieved by applying target values set in the manufacturer manual and using BC FlowSet Pro beads to adjust the voltages that enabled optimal detection and separation of dim and bright antigens. After pilot testing (data not shown), the NaviosTM instrument ClearLLab 10C settings were deemed appropriate for the DURAClone multicolour data acquisition and analysis.The NaviosTM process control  using four monoclonal antibodies, CD14 FITC, CD13 PE, CD45 PERCP and CD3 APC , was done.The FACSCalibur flow cytometer was used to acquire the predicate in-house second-line 2–4-colour fluorescence panels using CellQuest software . Quality control performed on the BD FACSCalibur included daily assessment of background contamination, carryover, acquisition, and analysis of manufacturer-recommended 3-colour and APC Calibrite beads . Further acquisition and analysis of Immunotrol3 and 0.0372% ethylenediaminetetraacetic acid; Merck, Darmstadt, Germany) in a 15 mL conical centrifuge tube for 15 min at room temperature. After incubation, samples were spun at 3000 g for 3 min, the supernatant was decanted, while the pellet was washed four times with 14 mL phosphate-buffered saline at pH 7.3 ± 2  containing 0.09% sodium azide (NaN3)  and 0.2% bovine serum albumin . Following washing, samples were reconstituted to 0.5 mL with phosphate-buffered saline, and the white cell count was determined. Cells were then diluted (or concentrated) to achieve the recommended cellular concentration of ≤ 10 000 cells/µL for monoclonal antibody incubation. Subsequently, 100 µL of this cell concentrate, which contained approximately 106 cells, was added to the ClearLLab 10C and in-house panels  with 14.5 mL isotonic ammonium chloride pH 7.1–7.4  events were collected. Paint-A-Gate software  was used for analysis of raw FACSCalibur flow cytometric data with a primary gating focus using CD45 and side scatter.23 First, doublets, debris and unlysed red blood cells were excluded, and, where possible, a neoplastic target population was identified in a primary gate using CD45 and side scatter. Thereafter, secondary gating focused on identifying the same target immunophenotype across each of the T, M1 and M2 panels.16 Finally, simultaneous identification of normal populations in the background was based on local in-house developed gating strategies to identify granulocytes, monocytes and mature lymphocytes using CD45, side scatter characteristics and specific regular expression of the markers studied  events were acquired on the Navios. Kaluza C™ version 1.1  software was used to analyse all raw listmode data to facilitate clinical interpretation. Kaluza C™ ClearLLab B, T, M1 and M2, and DURAClone panel analysis protocols were developed according to manufacturer specifications. studied .23 The relative intensity of expression of cell surface antigens was used to discriminate between normal and aberrant immunophenotypes according to manufacturer specifications. Positivity (or no expression) of individual markers was established, and overall haematolymphoid immunophenotypes sub-classified according to the established positive markers outlined in the World Health Organization (WHO) 2016 classification of tumours of haemopoietic and lymphoid tissues.24 Specifically, expression of CD27, CD43, CD56, CD58, and CD81, which were not included in the predicate ClearLLab 10C and in-house second-line panel analysis, were verified on background  populations as evidence of a satisfactory internal positive control and proof that the reagents met the manufacturer’s performance specifications. In the ALB analyses, typical secondary backbone markers, including co-expression of CD19/CD10/CD34 enabled further characterisation of markers CD20, CD38, and CD58 and established the presence of normal precursor B-cells (haematogones) or abnormal precursor B-cells. Likewise, for the DURAClone RE CLB, dual expression of either CD19/CD5 or CD19/CD20 was used to identify the ‘target’ or neoplastic B-cell population before specific characterisation of CD43, CD79b, CD81 and ROR1 expression. Plasma cells were defined in the DURAClone RE PC panel by the dual expression of both CD38 and CD138; a normal or malignant plasma cell immunophenotype was subsequently noted following interrogation of markers including CD81, CD27, CD19, CD200, CD56, and CD45.A similar approach was used for each of the comparative second-line DURAClone RE ALB, RE CLB, and RE PC listmode data analysis but additionally incorporated manufacturer-recommended gating strategies for clinical interpretation of data.Clinical diagnostic outcomes were collated into Microsoft Excel  spreadsheets. Marker expressions and specific clinical diagnoses noted for the DURAClone RE ALB, RE CLB, and RE PC panels were compared to the clinical diagnostic outcomes from the existing in-house 2–4-colour antibody panels described . The agrTwenty samples were tested with the DURAClone ALB panel. The DURAClone RE ALB evaluation on both routine diagnostic precursor B-ALL samples, and patient samples who were being followed up after therapy for precursor B-ALL, revealed 100% positive and negative agreement to ClearLLab 10C reported outcomes . The estn = 20). All CLL cases (n = 16/20) showed under-expression (weak) of CD81, CD79b and CD20 (n = 1) showed a bright expression of CD79b. Three cases (15%) evaluated could not be definitively sub-classified with the in-house second-line panel analysis or the DURAClone RE CLB reagents. An additional control sample  (n = 1) PCD had positive surface CD117 expression  . A singlpression  on the a16 fixed-tube, standardised, pre-titrated reagent system, ClearLLab 10C , and ClearLLab LS11 were recently implemented to replace our outdated FACSCalibur/Paint-a-gate method for routine immunophenotypic investigation of haematological neoplasms at our laboratory. This new system provides flow cytometric workup for haematological neoplasms and enables processing up to 400 samples a month. Previously, leukaemia flow cytometry testing was limited to simple 2–4-colour analysis; sample setup was manually intensive and error-prone. Systems previously implemented into our laboratory streamlined standardised and automated testing and improved workflow and quality of reporting.27 Similarly, implementing the ClearLLab 10C and LS system in our laboratory16 has markedly improved workflow and minimised errors while providing standardised and substantially better quality-controlled sample and data acquisition and comprehensive data. The latter allows for improved target population identification, especially for small populations.The recently evaluated,28 and could theoretically be assembled as required in the lab to provide additional marker panels needed to complete full patient immunophenotypic investigation. However, setting up quality-controlled multicolour panels in low- and middle-income countries is both challenging and less reliable16. The ClearLLab system facilitates detailed diagnosis across a broad range of acute leukaemias, and is sufficiently sensitive to detect the presence of a mature B-cell LPD, but it lacks specificity to discern and diagnose different mature B-cell LPD9 or confirm PCDs. For example, although the B-cell panel is useful to differentiate precursor B-cell from more mature B-cell LPD, the panel does not accommodate sufficient markers to discern different types of mature LPDs, including CD23, CD43, CD79b and FMC-7 markers necessary to distinguish B-cell LPD per the WHO classification.24 Likewise, markers, such as CD58, for diagnosis for subsequent follow-up of precursor B-ALL, or CD79a and CD81, would be of value in the B-cell panel to discern neoplasm from haematogones. Plasma cell dyscrasias are also not easily identified in the ClearLLab system, which lacks CD138 and other markers useful for diagnoses and follow-up of PCD.30 Although CD38, CD56, and CD45, used to identify plasma cells, are included in the ClearLLab panels, plasma cell identification is not definitive. Specifically, CD56, included in the T-cell panel, is assessed separately from CD38 (included in the B-cell and M2 panels). A pre-titrated-monoclonal panel including all three markers would suitably enhance the ClearLLab system; the DURAClone panels used in this study provided this supplement format.Complementary and supplementary multicolour panel options are published32 along with CD81 expression.31 CD58 in the index test ALB tube proved to be an excellent adjunct for assessing precursor B-cells and was helpful in discerning abnormal precursor B-cells from normal precursor B-cells (haematogones)31. Distinct discordant CD123 and CD34 expression patterns have also been described on haematogones and can be used in a strategy to determine B-ALL minimal residual disease.32 CD123 was not however included in the ALB tube. In our predicate method, the ClearLLab 10C system, co-expression of CD123 with CD34 and CD19 assessment within the ClearLLab M2 panel, used to identify precursor B-cells, together with CD19, CD10 and CD34 co-expression in the ClearLLab B-cell tube, therefore offers more diagnostic information than the DURAClone ALB tube alone for the follow-up of precursor B-ALL and residual disease assessment. The ClearLLab 10C system was developed to diagnose early and later B-cell lymphoproliferative disorders; these 10-colour panels are fixed. Thus, additional markers cannot be tested for in the panels. However, additional markers can be added to the RE ALB, fine-tuned toward detecting early B-cell precursors only. CD22 and CD123 could be valuable additions to the ALB panel. For example, a liquid reagent drop-in of CD22-APC in the free APC fluorescence channel of the DURAClone RE ALB panel could provide a baseline assessment of CD22 expression on B-ALL blasts, which would be useful for deciding on anti-CD22 targeted therapy in B-ALL patients. CD123 could also be added in a second available channel to enable further discrimination of disease (B-ALL) from normal haematogones  and clinical diagnostic outcomes agreement. The additional markers in the DURAClone RE CLB panel, including CD81, ROR1, CD79b and CD43 which are not included in the laboratory predicate ClearLLab system, specifically the B-cell tube, met the requirements for intended use and the manufacturer performance specifications. The DURAClone RE CLB panel proved to be an excellent fixed supplementary panel to replace our outdated second-line investigation for the workup of mature B-cell lymphoid proliferations and proved to be especially useful for discerning CD19/CD5 positive haematolymphoid disease subtypes. The surface CD81, CD79b and CD20 showed consistent under-expression in CLL, while the presence of ROR1 was also helpful in discerning CLL over mantle cell lymphoma as described in previous studies,33 especially when interpreted in conjunction with CD200 expression in the ClearLLab B-cell panel.35 In the event of a CD19/CD10 co-expressing mature B-cell lymphoid proliferation, CD43 in the DURAClone RE CLB panel was shown to be a useful cell surface marker to discriminate follicular lymphoma from a high-grade B-cell lymphoma. Similarly, CD79b within the DURAClone RE CLB panel, read together with CD38 expression in the ClearLLab B-cell tube, identifies a mature B-cell lymphoid proliferation with plasmacytoid differentiation. As for the ALB panel, free channels in the panel allow for additional flexibility to investigate related markers that are regarded as useful to discern mature B-cell haematolymphoid subtypes. A liquid reagent drop-in of CD23-ECD in the DURAClone RE CLB panel could further confirm B-cell CLL and discern B-cell CLL from other CD19/CD5 co-expressing target populations.33The expression of CD5, CD19, CD20 and CD45 in the DURAClone RE CLB reagent showed excellent cross-panel marker expression, equivalent to ClearLLab 10C B panel expression, with 100% (n = 17/17) clinical diagnostic concordance (agreement). In addition, as previously described,29 CD27 and CD8130 proved to be valuable markers to discern aberrant plasma cells over normal plasma cells. Finally, the usefulness of the RE PC panel can be further extended by adding CD117-ECD30 reagents. For one sample, we added CD117-ECD reagents to the PC tube, confirming the presence of malignant plasma cells and minimal residual disease assessment.14,30.The CD38, CD138, CD45, CD19, CD56, and CD200 expressions in the DURAClone RE PC panel showed excellent cross-panel marker expression and equivalent diagnostic outcome and FACSCalibur/PAG analysis outcomes, with 100% (23 (of the particular laboratory’s flow cytometer) can accommodate data collection from the fluorochromes used in the ClearLLab 10C or DURAClone panels.Firstly, the sample size per panel evaluated is small, and further studies are needed to confirm the outcomes reported here. Secondly, the fluorochromes used in the panels are specifically designed for use on a BC Navios instrument. Therefore, the products could be used on alternative instruments only if the respective filter setupsThis study confirms that the BC DURAClone ALB and CLB panels are suitable to provide additional second-line immunophenotypic workup of precursor B-ALL and mature B-cell lymphoproliferative disorders resepectively, at disease presentation and are suitable to supplement first-line ClearLLab 10C laboratory predicate method testing. The under-expression of CD81, CD79b, and positive CD43 and ROR1 expression, noted in the DURAClone CLB panel specifically assists in distinguishing B-cell CLL from mantle cell lymphoma and other CD5 negative B-cell LPDs.The DURAClone RE PC panel is suitable for the second-line investigation of PCD. CD27, CD56, CD81 and CD200, together with CD19, CD38 and CD138 in a single analysis, were efficient in identifying aberrant plasma cells.31 in the RE ALB tube may however be a valuable adjunct for discerning normal reactive B-cell precursors from the minimal residual disease at follow-up.Although there was diagnostic and individual marker concordance, we did not find the ALB more useful for diagnosing B-ALL over our existing ClearLLab system (utilising the B-cell and M2 tubes). The inclusion of CD58Lastly, the potential benefit of using commercialised multicolour lyophilised fixed panel preparations having stable, standardised antibody reagents reduces the risks of technical error, improves laboratory efficiency, and simplifies reagent inventory. In addition, the DURAClone ‘free fluorescence channels’ provide some additional flexibility for a laboratory to use their preferred markers to establish diseases (or not) of their choice.