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Anti-tumor efficacy of C4.4A-ADC in NSCLC PDX models in mice.

Supplementary Materials and Methods and Supplementary Figure Legends

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody–drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line–based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331–9. ©2016 AACR.

The anti-tumor efficacy of different FGFR2-ADC BAY 1187982 treatment schedules on MFM-223 human TNBC mouse model.

The expression of FGFR2 on tumor cell surface and half maximal inhibitory concentration of cell viability for FGFR2-ADC BAY 1187982 and control ADC.

Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.

Rogaratinib (BAY 1163877) is a highly potent and selective small-molecule pan-fibroblast growth factor receptor (FGFR) inhibitor (FGFR1-4) for oral application currently being investigated in phase 1 clinical trials for the treatment of cancer. In this publication, we report its discovery by de novo structure-based design and medicinal chemistry optimization together with its pharmacokinetic profile.

The first-in-class soluble guanylate cyclase (sGC) stimulator riociguat was recently introduced as a novel treatment option for pulmonary hypertension. Despite its outstanding pharmacological profile, application of riociguat in other cardiovascular indications is limited by its short half-life, necessitating a three times daily dosing regimen. In our efforts to further optimize the compound class, we have uncovered interesting structure-activity relationships and were able to decrease oxidative metabolism significantly. These studies resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY 1021189), currently in phase 3 trials for chronic heart failure, are now reported.

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody–drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line–based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331–9. ©2016 AACR.

Lead optimization of a high-throughput screening hit led to the rapid identification of aminopyrimidine ZK 304709, a multitargeted CDK and VEGF-R inhibitor that displayed a promising preclinical profile. Nevertheless, ZK 304709 failed in phase I studies due to dose-limited absorption and high inter-patient variability, which was attributed to limited aqueous solubility and off-target activity against carbonic anhydrases. Further lead optimization efforts to address the off-target activity profile finally resulted in the introduction of a sulfoximine group, which is still a rather unusual approach in medicinal chemistry. However, the sulfoximine series of compounds quickly revealed very interesting properties, culminating in the identification of the nanomolar pan-CDK inhibitor BAY 1000394, which is currently being investigated in phase I clinical trials.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

The number of cytotoxic payload classes with different modes-of-action which have been successfully employed in antibody-drug conjugates (ADC) is still rather limited. So far, only ADCs with microtubule inhibitors, DNA binding payloads or topoisomerase I inhibitors have been advanced into clinical testing. To this end, the identification of ADC payload classes with a novel mode of action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of kinesin spindle protein (KSP/Eg5) have generated interest due to their high antitumor potency. However, transferring the preclinical potency of small molecule KSP inhibitors (KSPis) into highly efficient clinical regimens with a sufficient therapeutic window has remained challenging. We have investigated a new pyrrole subclass of KSPis which showed subnanomolar potency against a large panel of tumor cell lines for their utility as a novel payload class in ADCs. Towards this goal different attachment sites for linkers have been explored in the KSPi molecule which were found compatible with cleavable and/or non-cleavable linkers. Subnanomolar potency and selectivity of ADCs with antibodies targeting either HER2, EGFR or TWEAKR could be demonstrated in vitro. For selected ADCs, the intracellular trafficking and metabolite formation was investigated and KSP inhibition was confirmed as the ADC mode of action. Depending on the linker composition differential profiles of the ADC metabolites with regard to efflux, cellular permeation, and bystander effect have been achieved. Moreover, specific accumulation in the tumor versus other tissues was demonstrated in biodistribution studies in vivo. In conclusion, KSP inhibitors have been established as a versatile new payload class for the generation of highly potent and selective ADCs. Citation Format: Hans-Georg Lerchen, Sven Wittrock, Nils Griebenow, Mario Lobell, Anne-Sophie Rebstock, Yolanda Cancho-Grande, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Anette Sommer, Sandra Berndt, Carsten Terjung, Heiner Apeler, Bertolt Kreft, Rolf Jautelat. Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2017-3234

Antibody-drug conjugates (ADCs) are promising agents that are developed for targeted delivery of cytotoxic payloads to tumor cells. ADCs share a common design of antibody, linker, and cytotoxic payload. Despite significant efforts, the number of available payload classes with a differentiated mode-of-action that can successfully be employed to generate antibody-drug conjugates (ADCs) is still rather limited. So far, only ADCs with microtubule depolymerizing or DNA binding payloads have been approved. The identification of ADC payload classes with a novel mode-of-action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of the kinesin spindle protein (KSP/Eg5/KIF11) have generated interest due to their high anti-tumor activity. However, the transfer of the potency of small molecule KSP inhibitors (KSPis) to highly efficient clinical regimens with a sufficient therapeutic window remains challenging. Through the conjugation of a novel pyrrole subclass of KSPis to antibodies targeting different cancer antigens, we generated a panel of ADCs and characterized them both in vitro and in vivo. ADCs targeting either EGFR or TWEAKR/Fn14 showed strong and specific internalization and displayed specific and potent anti-proliferative efficacy in vitro. In cytotoxicity assays, these ADCs exhibited sub-nanomolar potency in antigen-positive cancer cell lines (EGFR/TWEAKR-pos. NCI-H292; TWEAKR-pos. BxPC3, LoVo) and more than 100-fold selectivity versus non-targeted control-ADC containing the same linker and the same payload. Furthermore, selective anti-tumor efficacy of EGFR- and TWEAKR-KSPi-ADCs was demonstrated in vivo using both cancer cell line-derived models of NSCLC (NCI-H292), urothelial cell carcinoma (UCC) (KU-19-19), and renal cell carcinoma (A498), as well as in the TWEAKR-positive patient-derived xenograft UCC model BFX469. At doses of 5-10 mg/kg qw or bw potent anti-tumor efficacy with treated-to-control ratios (T/C) between 0.16 to 0.28 as well as complete regressions were observed. In summary, KSP inhibitors have been established as a promising new payload class allowing the generation of highly potent and selective ADCs for the treatment of solid tumors. Citation Format: Anette Sommer, Sandra Berndt, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Sven Wittrock, Anne-Sophie Rebstock, Lisa Dietz, Christoph Mahlert, Simone Greven, Nils Griebenow, Yolanda Cancho-Grande, Rolf Jautelat, Heiner Apeler, Bertolt Kreft. Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 46. doi:10.1158/1538-7445.AM2017-46

Aiming towards spiroketal-modified artificial cephalostatin molecules, two orthogonal approaches were investigated. First, the introduction of 17-O-functionality into hecogenin derivatives with a closed spiroketal moiety was accomplished by different remote-oxidation procedures. These allowed the synthesis of tetradecacyclic artificial cephalostatin molecules with improved tumor-inhibiting properties. Second, a novel reduction-oxidation pathway for spiroketal opening in sapogenins was discovered, which should provide the basis for a broad access towards spiroketal-modified building blocks for cephalostatins.

Trichodimerol (1) is a synthetically attractive natural product because of its potential medical use against septic shock and its striking molecular architecture. We report herein the possible biosynthetic pathway for its formation from the hexaketide sorbicillin (3) and our preliminary results towards the total synthesis of trichodimerol (1) and its congener demethyltrichodimerol (2). These studies provided a way to synthesize b-ketal ketones by a novel variation of the Mukaiyama Aldol reaction, afforded new insight into the mild and regioselective formation of silyl enol ethers, and allowed the preparation of the advanced intermediate 38. Furthermore, a number of unprecedented cascade reactions were discovered furnishing novel polycyclic, highly oxygenated compounds from simple starting materials.

The synthesis of the cephalostatin-analogous bis-steroidal pyrazines 6, 27a/b and 41 by the transformation of the C2-symmetrical diketone 6 as a central precursor, as well as the direct preparation of several non-symmetrical bis-steroidal pyrazines by coupling of enamino ketones (5, 40) with vinyl azides (17a/b) is reported. Furthermore, an improved procedure for preparation of the diketone 6 described earlier is presented.

Medizinische Chemikerinnen und Chemiker arbeiten bei Schering an der Generierung von neuen Wirkstoffen und sind somit initiale Schrittmacher der Medikamentenentwicklung.

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. Alterations in FGFR encoding genes are frequently observed in a variety of solid tumors including lung, gastric, breast and urothelial cancer. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients. BAY 1163877 is an orally active, highly potent and selective small molecule FGFR-1, -2 and -3 kinase inhibitor. We disclose for the very first time its discovery and chemical structure. BAY 1163877 was derived from a de novo structure-based design approach and medicinal chemistry optimization. Data on the structure activity relationship and the pharmacokinetic profile of the benzothiophenyl-pyrrolotriazine structure class will be presented. Based on its favorable preclinical profile, BAY 1163877 is currently being investigated in a Phase 1 clinical trial (NCT01976741). Citation Format: Marie-Pierre L. Collin, Mario Lobell, Walter Huebsch, Dirk Brohm, Mélanie Héroult, Klemens Lustig, Sylvia Gruenewald, Ulf Boemer, Rolf Jautelat, Holger Hess-Stump, Stefan Jaroch, Michael Brands, Karl Ziegelbauer. Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4332.

A seven-step cascade reaction-in which selective mesylation, epoxide formation, epoxide lysis, cyclization, reiterative oxidation, and nitrogen-oxygen exchange occur sequentially-facilitates the construction of the maleic anhydride moiety of CP molecules 1 and 2 (>93% yield per step). Unstable intermediates of this reaction sequence were detected, providing evidence for the proposed mechanism and resulting in the discovery of a new chemical entity.

X-ray structures from CDK2-aminopyrimidine inhibitor complexes led to the idea to stabilize the active conformation of aminopyrimidine inhibitors by incorporating the recognition site into a macrocyclic framework. A modular synthesis approach that relies on a new late-stage macrocyclization protocol that enables fast and efficient synthesis of macrocyclic aminopyrimidines was developed. A set of structurally diverse derivatives was prepared. Macrocyclic aminopyrimidines were shown to be multitarget inhibitors of CDK1/2 and VEGF-RTKs. In addition, potent antiproliferative activities toward various human tumor cells and a human tumor xenograft model were demonstrated.

Starting off in the early 90's the field of cephalostatin analogues has continually expanded over the last 10 years. First syntheses prepared symmetric analogues like 14b (119) and 26 (65), which were subsequently desymmetrized to provide analogues like beta-hydroxy ketone 31 (19). Importantly the straightforward approach provided already compounds with mu-molar potency and the same pattern of activity as cephalostatin 1 (1) (see Chapter 2.1). Chemically more demanding, two new methods for the directed synthesis of (bissteroidal) pyrazines were devised and subsequently applied to a wide variety of differently functionalized coupling partners. These new methods allowed for the synthesis of various analogues (Chapter 2.2.; and, last but not least, for the totals synthesis of several cephalostatin natural products; Chapter 1.). Functionalization and derivatization of the 12-position was performed (Chapter 2.1 and 3) and synthetic approaches to establish the D-ring double bond were successfully investigated (Chapter 3). [figure: see text] Dealing synthetically with the spiroketal moiety, novel oxidative opening procedures on monomeric delta 14, 15-steroids were devised as well as intensive studies regarding spiroketal synthesis and spiroketal rearrangements were conducted (Chapter 3.2. and 4.). Last but not least direct chemical modification of ritterazines and cephalostatins were studied, which provided a limited number of ritterazine analogues (Chapter 4.). All these synthetic activities towards analogues are summarized in Fig. 18. During this period of time the growing number of cephalostatins and ritterazines on the one hand and of analogues on the other hand provided several SAR trends, which can guide future analogue synthesis. The combined SAR findings are displayed in Fig. 19. So far it is apparent that: Additional methoxylations or hydroxylations in the steroidal A ring core structure (1-position) are slightly decreasing activity (compare cephalostatin 1 1 to cephalostatins 18, 19, 10, and 11). Not investigated by preparation of analogues. Additional hydroxylations in the B-ring (7- and 9-position) do not have a strong effect. They appear to decrease slightly the activity in the case of 9-position (compare cephalostatin 1 1 to cephalostatin 4) and are neutral in the case of the 7-position (compare ritterazines J and K). Analogue synthesis confirmed this: 7-ring-hydroxylation has little impact on activity, e.g. 109a (Table 6). C'-ring aryl compounds with a 12,17 connected spiroketal area are much less active (cephalostatins 5 and 6), meaning South 6 moiety reduces activity [figure: see text] Confirmed by analogue synthesis, e.g. 190a and 190b (Table 9). Regarding 12-functionalization it is apparent, that all cephalostatins/ritterazines possess either a free hydroxy or a keto function at this position (exemption: cephalostatins 5 and 6--very low activity). However, it is not apparent whether a 12,12'-diol or a 12-keto-12'-ol is favored. In the cephalostatin series the most potent compounds possess a 12-keto-12'-ol function, while in the ritterazine series the direct comparison of ritterazine B and ritterazine H clearly favors the 12,12'-diol setting. Synthesis of simple analogues like 31 showed a "cephalostatin trend" for favoring the 12-keto, 12'-alcohol functionalization. Synthesis of a cephalostatin 1-12'-alcohol 1a supported that trend (2 fold drop in activity). Synthesis of acylated ritterazine B derivatives proved that free hydroxy groups in 12-position are necessary for high activity. At least one 14,15-double bond is part of all highly active cephalostatins/ritterazines. All ritterazines lacking this feature display only low potency (but most of them possess the unfavorable North A moiety or have unfavorable combinations of moieties; vide infra). However, the 14,15-double bond may be necessary "only" for stereochemical reasons creating a specific "curvature" of the molecule by "bending" the D-ring down (for an in depth discussion on this topic: see Chapter 3). In line with this are the observations that 14,15-alpha-epoxides do substantially decrease activity (cephalostatins 14 and 15) while a 14,15-beta-epoxide does not decrease activity (cephalostatin 4). Also in line with the "curvature theory" is the fact that ritterazine B (14-beta-hydrogen) is even more potent than ritterazine G (14,15-double bond). Therefore it is not clear if--at least one--14,15-double bond is essential for high activity. The synthesis and biological evaluation of completely 14-beta-saturated analogues (like 14'-beta-hydrogen ritterazine B) could answer this question. Synthesis of the partially saturated analogues 14' alpha-cephalostatin 1 1c and 7-deoxy-14' alpha-ritterazine B 2a showed that the stronger the divergence of conformation implied by the saturation is, the higher is the loss of activity, thus underlining the "curvature hypothesis". Synthesis showed, that analogues possessing the 14,15-double bond(s) are substantially better soluble, e.g. 26. Furthermore, the D-Ring area turned out to be sensitive for modifications, since substantially differing analogues, like 162, 163, and 164 were completely inactive. At least one 17-hydroxy group is part of all highly active cephalostatins/ritterazines. Loss of one out of two 17-hydroxy groups does not decrease activity (compare ritterazine K and L) but of the second 17-hydroxy groups (along with the 7-hydroxy group) as seen in the ritterazine series (compare ritterazines A/T and B/Y) leads to a significant decrease in activity. Increased activity of 17-ether analogues 178 and 179 points into the same direction All highly active cephalostatins and ritterazines are substantially asymmetric. Cephalostatins and ritterazines that are symmetric--either consisting of two polar units (cephalostatin 12 and ritterazine K) or two unpolar units (ritterazine N and ritterazine R)--or almost symmetric (cephalostatin 13 and ritterazine J, L, M, O, S) show substantially diminished potency. However, one has to keep in mind, that even some of the symmetrical compounds (e.g. ritterazine K--96 nM in the NCI panel) still show strong cytostatic properties. Same trend was identified with simple analogues, e.g. compare 26 to 31. In addition to the basic requirement of overall substantial asymmetry for high activity there appears to be the necessity for a "polarity match" between both steroidal units (33)--as one has to be substantially more polar (high hydroxylation grade) than the other. (e.g. cephalostatin 1 (1): North 1--high hydroxylation grade--and South 1--low hydroxylation grade; or: ritterazine B (2): South 7--medium hydroxylation grade--and North G--very low hydroxylation grade). Not directly confirmed by Analogue Synthesis--some "polarity matched analogues" did not show appropriate activity, e.g. 198 and 197. 4 core moieties are privileged, meaning all highly active ritterazines/cephalostatins (see table 1) are constructed out of them. Namely these are North 1, South 1, South 7 and North G. Numerous analogues were prepared to probe questions regarding the mechanism of action of the cephalostatins, e.g. close cephalostatin analogues like 197 and 198 (70) with increased energy content in the spiroketal. However, so far the mechanism and mode of action of the cephalostatins remains unknown. In the absence of any structural information of the biological target(s), the understanding about the structural necessities for high cytostatic activity is still limited and thus the rational design of more simple, yet highly active analogues seems at the current stage elusive. Additionally, there are many open questions, e.g. how the "monomeric" OSW-1 (3) relates to the "dimeric" cephalostatins. It remains the hope that forthcoming studies will bring light into this so far nebulous area--enabling chemists in the long run to provide highly active analogues in substantial amounts for advanced pharmacological studies. In conclusion one can state that the first decade after the extraordinarily complex cephalostatin 1 (1) entered the scene was necessary for the chemists to explore novel ways towards cephalostatins and cephalostatin analogues. They have provided methods to prepare basically every thinkable cephalostatin analogue, have delivered simple analogues (< 10 steps) with substantial activity and shaped first SAR trends in the class of cephalostatins. Now the time has come for chemists to harvest the fruits of their long and enduring synthetic ventures by aiming towards highly active, yet still not too complex analogues, which could be available in substantial amounts for advanced pharmacological studies. And for pharmacologists to explore the therapeutic potential of the cephalostatins along with elucidation of the unknown mechanism. Clearly, there is much more to expect of the cephalostatins in the coming years.

Starting off in the early 90's the field of cephalostatin analogues has continually expanded over the last 10 years. First syntheses prepared symmetric analogues like 14b (119) and 26 (65), which were subsequently desymmetrized to provide analogues like beta-hydroxy ketone 31 (19). Importantly the straightforward approach provided already compounds with mu-molar potency and the same pattern of activity as cephalostatin 1 (1) (see Chapter 2.1). Chemically more demanding, two new methods for the directed synthesis of (bissteroidal) pyrazines were devised and subsequently applied to a wide variety of differently functionalized coupling partners. These new methods allowed for the synthesis of various analogues (Chapter 2.2.; and, last but not least, for the totals synthesis of several cephalostatin natural products; Chapter 1.). Functionalization and derivatization of the 12-position was performed (Chapter 2.1 and 3) and synthetic approaches to establish the D-ring double bond were successfully investigated (Chapter 3). [figure: see text] Dealing synthetically with the spiroketal moiety, novel oxidative opening procedures on monomeric delta 14, 15-steroids were devised as well as intensive studies regarding spiroketal synthesis and spiroketal rearrangements were conducted (Chapter 3.2. and 4.). Last but not least direct chemical modification of ritterazines and cephalostatins were studied, which provided a limited number of ritterazine analogues (Chapter 4.). All these synthetic activities towards analogues are summarized in Fig. 18. During this period of time the growing number of cephalostatins and ritterazines on the one hand and of analogues on the other hand provided several SAR trends, which can guide future analogue synthesis. The combined SAR findings are displayed in Fig. 19. So far it is apparent that: Additional methoxylations or hydroxylations in the steroidal A ring core structure (1-position) are slightly decreasing activity (compare cephalostatin 1 1 to cephalostatins 18, 19, 10, and 11). Not investigated by preparation of analogues. Additional hydroxylations in the B-ring (7- and 9-position) do not have a strong effect. They appear to decrease slightly the activity in the case of 9-position (compare cephalostatin 1 1 to cephalostatin 4) and are neutral in the case of the 7-position (compare ritterazines J and K). Analogue synthesis confirmed this: 7-ring-hydroxylation has little impact on activity, e.g. 109a (Table 6). C'-ring aryl compounds with a 12,17 connected spiroketal area are much less active (cephalostatins 5 and 6), meaning South 6 moiety reduces activity [figure: see text] Confirmed by analogue synthesis, e.g. 190a and 190b (Table 9). Regarding 12-functionalization it is apparent, that all cephalostatins/ritterazines possess either a free hydroxy or a keto function at this position (exemption: cephalostatins 5 and 6--very low activity). However, it is not apparent whether a 12,12'-diol or a 12-keto-12'-ol is favored. In the cephalostatin series the most potent compounds possess a 12-keto-12'-ol function, while in the ritterazine series the direct comparison of ritterazine B and ritterazine H clearly favors the 12,12'-diol setting. Synthesis of simple analogues like 31 showed a "cephalostatin trend" for favoring the 12-keto, 12'-alcohol functionalization. Synthesis of a cephalostatin 1-12'-alcohol 1a supported that trend (2 fold drop in activity). Synthesis of acylated ritterazine B derivatives proved that free hydroxy groups in 12-position are necessary for high activity. At least one 14,15-double bond is part of all highly active cephalostatins/ritterazines. All ritterazines lacking this feature display only low potency (but most of them possess the unfavorable North A moiety or have unfavorable combinations of moieties; vide infra). However, the 14,15-double bond may be necessary "only" for stereochemical reasons creating a specific "curvature" of the molecule by "bending" the D-ring down (for an in depth discussion on this topic: see Chapter 3). In line with this are the observations that 14,15-alpha-epoxides do substantially decrease activity (cephalostatins 14 and 15) while a 14,15-beta-epoxide does not decrease activity (cephalostatin 4). Also in line with the "curvature theory" is the fact that ritterazine B (14-beta-hydrogen) is even more potent than ritterazine G (14,15-double bond). Therefore it is not clear if--at least one--14,15-double bond is essential for high activity. The synthesis and biological evaluation of completely 14-beta-saturated analogues (like 14'-beta-hydrogen ritterazine B) could answer this question. Synthesis of the partially saturated analogues 14' alpha-cephalostatin 1 1c and 7-deoxy-14' alpha-ritterazine B 2a showed that the stronger the divergence of conformation implied by the saturation is, the higher is the loss of activity, thus underlining the "curvature hypothesis". Synthesis showed, that analogues possessing the 14,15-double bond(s) are substantially better soluble, e.g. 26. Furthermore, the D-Ring area turned out to be sensitive for modifications, since substantially differing analogues, like 162, 163, and 164 were completely inactive. At least one 17-hydroxy group is part of all highly active cephalostatins/ritterazines. Loss of one out of two 17-hydroxy groups does not decrease activity (compare ritterazine K and L) but of the second 17-hydroxy groups (along with the 7-hydroxy group) as seen in the ritterazine series (compare ritterazines A/T and B/Y) leads to a significant decrease in activity. Increased activity of 17-ether analogues 178 and 179 points into the same direction All highly active cephalostatins and ritterazines are substantially asymmetric. Cephalostatins and ritterazines that are symmetric--either consisting of two polar units (cephalostatin 12 and ritterazine K) or two unpolar units (ritterazine N and ritterazine R)--or almost symmetric (cephalostatin 13 and ritterazine J, L, M, O, S) show substantially diminished potency. However, one has to keep in mind, that even some of the symmetrical compounds (e.g. ritterazine K--96 nM in the NCI panel) still show strong cytostatic properties. Same trend was identified with simple analogues, e.g. compare 26 to 31. In addition to the basic requirement of overall substantial asymmetry for high activity there appears to be the necessity for a "polarity match" between both steroidal units (33)--as one has to be substantially more polar (high hydroxylation grade) than the other. (e.g. cephalostatin 1 (1): North 1--high hydroxylation grade--and South 1--low hydroxylation grade; or: ritterazine B (2): South 7--medium hydroxylation grade--and North G--very low hydroxylation grade). Not directly confirmed by Analogue Synthesis--some "polarity matched analogues" did not show appropriate activity, e.g. 198 and 197. 4 core moieties are privileged, meaning all highly active ritterazines/cephalostatins (see table 1) are constructed out of them. Namely these are North 1, South 1, South 7 and North G. Numerous analogues were prepared to probe questions regarding the mechanism of action of the cephalostatins, e.g. close cephalostatin analogues like 197 and 198 (70) with increased energy content in the spiroketal. However, so far the mechanism and mode of action of the cephalostatins remains unknown. In the absence of any structural information of the biological target(s), the understanding about the structural necessities for high cytostatic activity is still limited and thus the rational design of more simple, yet highly active analogues seems at the current stage elusive. Additionally, there are many open questions, e.g. how the "monomeric" OSW-1 (3) relates to the "dimeric" cephalostatins. It remains the hope that forthcoming studies will bring light into this so far nebulous area--enabling chemists in the long run to provide highly active analogues in substantial amounts for advanced pharmacological studies. In conclusion one can state that the first decade after the extraordinarily complex cephalostatin 1 (1) entered the scene was necessary for the chemists to explore novel ways towards cephalostatins and cephalostatin analogues. They have provided methods to prepare basically every thinkable cephalostatin analogue, have delivered simple analogues (10 steps) with substantial activity and shaped first SAR trends in the class of cephalostatins. Now the time has come for chemists to harvest the fruits of their long and enduring synthetic ventures by aiming towards highly active, yet still not too complex analogues, which could be available in substantial amounts for advanced pharmacological studies. And for pharmacologists to explore the therapeutic potential of the cephalostatins along with elucidation of the unknown mechanism. Clearly, there is much more to expect of the cephalostatins in the coming years.

Starting off in the early 90's the field of cephalostatin analogues has continually expanded over the last 10 years. First syntheses prepared symmetric analogues like 14b (119) and 26 (65), which were subsequently desymmetrized to provide analogues like beta-hydroxy ketone 31 (19). Importantly the straightforward approach provided already compounds with mu-molar potency and the same pattern of activity as cephalostatin 1 (1) (see Chapter 2.1). Chemically more demanding, two new methods for the directed synthesis of (bissteroidal) pyrazines were devised and subsequently applied to a wide variety of differently functionalized coupling partners. These new methods allowed for the synthesis of various analogues (Chapter 2.2.; and, last but not least, for the totals synthesis of several cephalostatin natural products; Chapter 1.). Functionalization and derivatization of the 12-position was performed (Chapter 2.1 and 3) and synthetic approaches to establish the D-ring double bond were successfully investigated (Chapter 3). [figure: see text] Dealing synthetically with the spiroketal moiety, novel oxidative opening procedures on monomeric delta 14, 15-steroids were devised as well as intensive studies regarding spiroketal synthesis and spiroketal rearrangements were conducted (Chapter 3.2. and 4.). Last but not least direct chemical modification of ritterazines and cephalostatins were studied, which provided a limited number of ritterazine analogues (Chapter 4.). All these synthetic activities towards analogues are summarized in Fig. 18. During this period of time the growing number of cephalostatins and ritterazines on the one hand and of analogues on the other hand provided several SAR trends, which can guide future analogue synthesis. The combined SAR findings are displayed in Fig. 19. So far it is apparent that: Additional methoxylations or hydroxylations in the steroidal A ring core structure (1-position) are slightly decreasing activity (compare cephalostatin 1 1 to cephalostatins 18, 19, 10, and 11). Not investigated by preparation of analogues. Additional hydroxylations in the B-ring (7- and 9-position) do not have a strong effect. They appear to decrease slightly the activity in the case of 9-position (compare cephalostatin 1 1 to cephalostatin 4) and are neutral in the case of the 7-position (compare ritterazines J and K). Analogue synthesis confirmed this: 7-ring-hydroxylation has little impact on activity, e.g. 109a (Table 6). C'-ring aryl compounds with a 12,17 connected spiroketal area are much less active (cephalostatins 5 and 6), meaning South 6 moiety reduces activity [figure: see text] Confirmed by analogue synthesis, e.g. 190a and 190b (Table 9). Regarding 12-functionalization it is apparent, that all cephalostatins/ritterazines possess either a free hydroxy or a keto function at this position (exemption: cephalostatins 5 and 6--very low activity). However, it is not apparent whether a 12,12'-diol or a 12-keto-12'-ol is favored. In the cephalostatin series the most potent compounds possess a 12-keto-12'-ol function, while in the ritterazine series the direct comparison of ritterazine B and ritterazine H clearly favors the 12,12'-diol setting. Synthesis of simple analogues like 31 showed a "cephalostatin trend" for favoring the 12-keto, 12'-alcohol functionalization. Synthesis of a cephalostatin 1-12'-alcohol 1a supported that trend (2 fold drop in activity). Synthesis of acylated ritterazine B derivatives proved that free hydroxy groups in 12-position are necessary for high activity. At least one 14,15-double bond is part of all highly active cephalostatins/ritterazines. All ritterazines lacking this feature display only low potency (but most of them possess the unfavorable North A moiety or have unfavorable combinations of moieties; vide infra). However, the 14,15-double bond may be necessary "only" for stereochemical reasons creating a specific "curvature" of the molecule by "bending" the D-ring down (for an in depth discussion on this topic: see Chapter 3). In line with this are the observations that 14,15-alpha-epoxides do substantially decrease activity (cephalostatins 14 and 15) while a 14,15-beta-epoxide does not decrease activity (cephalostatin 4). Also in line with the "curvature theory" is the fact that ritterazine B (14-beta-hydrogen) is even more potent than ritterazine G (14,15-double bond). Therefore it is not clear if--at least one--14,15-double bond is essential for high activity. The synthesis and biological evaluation of completely 14-beta-saturated analogues (like 14'-beta-hydrogen ritterazine B) could answer this question. Synthesis of the partially saturated analogues 14' alpha-cephalostatin 1 1c and 7-deoxy-14' alpha-ritterazine B 2a showed that the stronger the divergence of conformation implied by the saturation is, the higher is the loss of activity, thus underlining the "curvature hypothesis". Synthesis showed, that analogues possessing the 14,15-double bond(s) are substantially better soluble, e.g. 26. Furthermore, the D-Ring area turned out to be sensitive for modifications, since substantially differing analogues, like 162, 163, and 164 were completely inactive. At least one 17-hydroxy group is part of all highly active cephalostatins/ritterazines. Loss of one out of two 17-hydroxy groups does not decrease activity (compare ritterazine K and L) but of the second 17-hydroxy groups (along with the 7-hydroxy group) as seen in the ritterazine series (compare ritterazines A/T and B/Y) leads to a significant decrease in activity. Increased activity of 17-ether analogues 178 and 179 points into the same direction All highly active cephalostatins and ritterazines are substantially asymmetric. Cephalostatins and ritterazines that are symmetric--either consisting of two polar units (cephalostatin 12 and ritterazine K) or two unpolar units (ritterazine N and ritterazine R)--or almost symmetric (cephalostatin 13 and ritterazine J, L, M, O, S) show substantially diminished potency. However, one has to keep in mind, that even some of the symmetrical compounds (e.g. ritterazine K--96 nM in the NCI panel) still show strong cytostatic properties. Same trend was identified with simple analogues, e.g. compare 26 to 31. In addition to the basic requirement of overall substantial asymmetry for high activity there appears to be the necessity for a "polarity match" between both steroidal units (33)--as one has to be substantially more polar (high hydroxylation grade) than the other. (e.g. cephalostatin 1 (1): North 1--high hydroxylation grade--and South 1--low hydroxylation grade; or: ritterazine B (2): South 7--medium hydroxylation grade--and North G--very low hydroxylation grade). Not directly confirmed by Analogue Synthesis--some "polarity matched analogues" did not show appropriate activity, e.g. 198 and 197. 4 core moieties are privileged, meaning all highly active ritterazines/cephalostatins (see table 1) are constructed out of them. Namely these are North 1, South 1, South 7 and North G. Numerous analogues were prepared to probe questions regarding the mechanism of action of the cephalostatins, e.g. close cephalostatin analogues like 197 and 198 (70) with increased energy content in the spiroketal. However, so far the mechanism and mode of action of the cephalostatins remains unknown. In the absence of any structural information of the biological target(s), the understanding about the structural necessities for high cytostatic activity is still limited and thus the rational design of more simple, yet highly active analogues seems at the current stage elusive. Additionally, there are many open questions, e.g. how the "monomeric" OSW-1 (3) relates to the "dimeric" cephalostatins. It remains the hope that forthcoming studies will bring light into this so far nebulous area--enabling chemists in the long run to provide highly active analogues in substantial amounts for advanced pharmacological studies. In conclusion one can state that the first decade after the extraordinarily complex cephalostatin 1 (1) entered the scene was necessary for the chemists to explore novel ways towards cephalostatins and cephalostatin analogues. They have provided methods to prepare basically every thinkable cephalostatin analogue, have delivered simple analogues (< 10 steps) with substantial activity and shaped first SAR trends in the class of cephalostatins. Now the time has come for chemists to harvest the fruits of their long and enduring synthetic ventures by aiming towards highly active, yet still not too complex analogues, which could be available in substantial amounts for advanced pharmacological studies. And for pharmacologists to explore the therapeutic potential of the cephalostatins along with elucidation of the unknown mechanism. Clearly, there is much more to expect of the cephalostatins in the coming years.

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. FGF signaling has been demonstrated to be altered in a high proportion of cancers, with activating mutations and/or overexpression of FGFRs frequently observed in lung, gastric, breast and urothelial tumors. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients. BAY 1163877 is as an orally available, selective and potent inhibitor of FGFR-1, -2 and -3 kinase activity. BAY 1163877 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. BAY 1163877 inhibited FGFR-1, -2, -3 kinase activity in the nanomolar range and demonstrated a kinase selectivity profile for FGFR-1, -2 and -3 over 222 kinases tested. BAY 1163877 inhibited proliferation of various cancer cell lines in vitro and phosphorylation of downstream signaling molecules. BAY 1163877 was also tested in vivo in monotherapy and combination therapy on various human xenografts and syngeneic tumors and inhibited growth of tumors presenting at least one FGFR alteration.Overall, the in vitro and in vivo studies confirm that the FGFR inhibitor BAY 1163877 is a potent and selective inhibitor of altered FGFRs pathways in cancer models. A Phase 1 clinical trial (NCT01976741) has been initiated. Citation Format: Melanie Heroult, Peter Ellinghaus, Christian Sieg, Dirk Brohm, Sylvia Gruenewald, Marie-Pierre Collin, Ulf Boemer, Mario Lobell, Walter Huebsch, Matthias Ocker, Stuart Ince, Andrea Haegebarth, Rolf Jautelat, Holger Hess-Stumpp, Michael Brands, Karl Ziegelbauer. Preclinical profile of BAY 1163877 - a selective pan-FGFR inhibitor in phase 1 clinical trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2014-1739

C4.4a (LYPD3) has been identified previously as a cancer- and metastasis-associated internalizing cell surface protein. Targeting C4.4a with a specific antibody-drug conjugate (ADC) represents an unique opportunity to treat tumors with high unmet medical need such as squamous cell carcinomas SCC, in particular lung SCC. We have generated an anti-C4.4a ADC consisting of a fully human monoclonal antibody linked to a non cell-permeable tubulin-binding auristatin cytotoxic agent (technology licensed from Seattle Genetics). In vitro, anti-C4.4a ADC showed an anti-proliferative efficacy (IC50) in the nanomolar range in cell lines endogenously expressing C4.4a (e.g. human lung cancer cell lines NCI-H292 and NCI-H322). High ADC stability and selectivity was observed in transfected A549 lung cancer cells over-expressing C4.4a compared to mock-transfected cells. In vivo, anti-C4.4a ADC exhibited a potent and selective antitumor activity in various human xenograft models (NCI-H292, NCI-H322, SCC-4) as well as in two SCC (Lu7433, Lu7343) and one pleomorphic (Lu7064) patient-derived lung cancer xenograft models. The in vivo efficacy is strictly target-dependent and selective as no efficacy was observed in C4.4a negative models (Fadu, Lu 7700) or using a non-specific isotype antibody ADC (NCI-H292, NCI-H322). A minimal effective dose (MED) as low as 1.9 mg/kg, response rates of up to 100%, and additive anti-tumor efficacy in combination with cisplatin were observed in the NCI-H292 xenograft model. Furthermore, it has been demonstrated that NCI-H292 were still sensitive to ADC treatment when tumors were allowed to regrow after the initial treatment cycle(). The anti-C4.4a ADC, which is fully cross-reactive with the mouse orthologue of C4.4a, was well tolerated at efficacious doses. Reversible skin reddening was observed only at doses markedly higher than the MED. In summary, anti-C4.4a ADC is a promising therapeutic candidate for the treatment of C4.4a-expressing squamous cell carcinomas, andpreclinical development has been initiated. Citation Format: Joerg Willuda, Lars Linden, Hans-Georg Lerchen, Charlotte Kopitz, Sven Golfier, Ute Bach, Joachim Schumacher, Beatrix Stelte-Ludwig, Oliver Von Ahsen, Claudia Schneider, Frank Dittmer, Rudolf Beier, Sherif El-Sheik, Jan Tebbe, Gabriele Leder, Heiner Apeler, Rolf Jautelat, Bertolt Kreft, Karl Ziegelbauer. Preclinical anti-tumor efficacy of an anti-C4.4a (LYPD3) antibody drug conjugate for the treatment of lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2014-5445

A seven-step cascade reaction-in which selective mesylation, epoxide formation, epoxide lysis, cyclization, reiterative oxidation, and nitrogen-oxygen exchange occur sequentially-facilitates the construction of the maleic anhydride moiety of CP molecules 1 and 2 (>93% yield per step). Unstable intermediates of this reaction sequence were detected, providing evidence for the proposed mechanism and resulting in the discovery of a new chemical entity.