Bisphosphonate-Based Conjugates and Derivatives As Potential Therapeutic Agents in Osteoporosis, Bone Cancer and Metastatic Bone Cancer

International Journal of Molecular Sciences

Review Bisphosphonate-Based Conjugates and Derivatives as Potential Therapeutic Agents in Osteoporosis, Bone Cancer and Metastatic Bone Cancer

Zintle Mbese and Blessing A. Aderibigbe *

Department of Chemistry, Alice Campus, University of Fort Hare, Alice 5700, South Africa; [email protected] * Correspondence: [email protected]; Tel.: +27-(0)-406-022-266; Fax: +086-662-8802

Abstract: Metastatic bone cancer occurs in every type of cancer but is prevalent in lung, breast, and prostate cancers. These metastases can cause extensive morbidity, including a range of skeletal-related events, often painful and linked with substantial hospital resource usage. The treatment used is a combination of chemotherapy and surgery. However, anticancer drugs are still limited due to severe side effects, drug resistance, poor blood supply, and non-speciﬁc drug uptake, necessitating high toxic doses. Bisphosphonates are the main class of drugs utilized to inhibit metastatic bone cancer. It is also used for the treatment of osteoporosis and other bone diseases. However, bisphosphonate also suffers from serious side effects. Thus, there is a serious need to develop bisphosphonate conjugates with promising therapeutic outcomes for treating metastatic bone cancer and osteoporosis. This review article focuses on the biological outcomes of designed bisphosphonate-based conjugates for

the treatment of metastatic bone cancer and osteoporosis. Keywords: osteoporosis; bisphosphonates; bone cancer; metastatic cancer Citation: Mbese, Z.; Aderibigbe, B.A. Bisphosphonate-Based Conjugates and Derivatives as Potential Therapeutic Agents in Osteoporosis, Bone Cancer and Metastatic Bone 1. Introduction Cancer. Int. J. Mol. Sci. 2021, 22, 6869. Bisphosphonates (BPs) are a significant class of medications that are utilized in the https://doi.org/10.3390/ treatment of diseases linked with low bone mass, including Paget’s disease, osteoporosis, ijms22136869 and the inhibition of skeletal-related events (SRE) in individuals with osteolytic bone metastases and multiple myeloma [1,2]. BP was synthesized for the first time in the 1800s. Academic Editor: Manuel Scimeca Its structure in Figure1 is composed of two phosphate groups binding to a carbon atom (P-C-P), and their potential to bind to the bone surface is dependent on the P-C-P bond. Received: 9 March 2021 This bond also improves their binding to the mineralized bone matrix with a subsequent Accepted: 20 April 2021 inhibitory effect on bone resorption [3]. The R1 side chain that contains a hydroxyl group Published: 26 June 2021 (OH) is attached to the central carbon atom and further enhances its binding affinity [1]. BPs anti-resorptive activity is ascribed to the R2 side chain. The existence of a primary Publisher’s Note: MDPI stays neutral amine (NH2) that is attached to the central carbon atom raises strongly anti-resorptive with regard to jurisdictional claims in effectiveness [1]. Hence, the addition of hydroxyl group and/or primary amine group published maps and institutional affil- increases the affinity for calcium ions, which results in favored localization of the remedies iations. to the sites of bone remodeling [4]. BPs are classified into two groups which are nitrogen- containing bisphosphonates (N-BPs) e.g., pamidronate, neridronate, alendronate, etc., and non-nitrogen containing bisphosphonates (N-NBPs) e.g., etidronate and clodronate [5]. They are also classified into three generations based on their therapeutic effects, as shown in Copyright: © 2021 by the authors. Table1. The 3rd generation BPs are more effective than the 1st and 2nd generation BPs [ 5,6]. Licensee MDPI, Basel, Switzerland. The N-NBPs are incorporated into the energy pathways of the osteoclast, resulting in This article is an open access article disrupted cellular energy metabolism leading to apoptosis. N-BPs exert their effect on distributed under the terms and osteoclasts via their inhibition of the mevalonate pathways, resulting in the disruption of conditions of the Creative Commons the intracellular signaling and induction of apoptosis [7]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Int. J. Mol. Sci. 2021, 22, 6869. https://doi.org/10.3390/ijms22136869 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 23

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 23 Int. J. Mol. Sci. 2021, 22, 6869 2 of 21 OH R1 O

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWOP OHC P R1 OH O 2 of 23

OH R2 OH OH R1 O OP1 C P OH Int. J. Mol. Sci.Int. 2021 J. Mol., 22 Sci., x FOR 2021 PEER, 22, x REVIEW FOR PEER REVIEW 2 of 23 2 of 23 Int. J. Mol. Sci.Int. 2021 J. Mol., 22, xSci. FOR 2021 PEER, 22, REVIEWx FOR PEER REVIEW 2 of 23 2 of 23 Int. J. Mol. Sci.Int. 2021 J. Mol., 22, xSci. FOR 2021 PEER, 22, REVIEWx FOR PEER REVIEW 2 of 23 2 of 23

FigureOP 1. StructureC of Bisphosphonate.P OH

OH R1 OH O R1 O Table 1. The structuresOH R1 OH of 3O generations2R1 O of bisphosphonates. OHOH R1 OH OR R1 O OH OH R2 OH

1st Generation OPOPC P C OH2ndP GenerationOH 3rd Generation OPOPC P C OH P OH OPOPH2NC P C OH P OH 1 1 OH R2 OH OHR2 OH 2 2 OH CH3 O Figure 1. StructureOH ofR Bisphosphonate.OH OH R OH N Figure 1. StructureOH R of2 OH Bisphosphonate.OH R2 OH Figure 1.1 Structure1 of Bisphosphonate. 1 1 Table 1. The structures1 of 3 generations1 of bisphosphonates. Figure Table1. StructureFigure 1. 1.The of Structure Bisphosphonate. structures of Bisphosphonate. of 3 generations of bisphosphonates. N OPC P OHFigure 1. StructureFigure 1.of Structure Bisphosphonate. of Bisphosphonate. Figure 1. StructureFigure 1.of Structure Bisphosphonate.OH of Bisphosphonate. O OH 1st Generation Table 1. TheTable structures 1. The structuresof 3 generations of2nd 3 generations of Generation bisphosphonates. of bisphosphonates. 3rd Generation O 1st GenerationTableTable 1. 1. The TheTable structures 1.structures The of 2nd structures 3 generations Generation of 3of generationsof bisphosphonates.3 generations of bisphosphonates. of bisphosphonates. 3rd Generation Table 1. TheTable structuresH2N 1. The of structures 3 generations of 3 generationsof bisphosphonates. of bisphosphonates. 1st Generation1st Generation 2nd Generation2nd Generation 3rd Generation3rd Generation OH OH OH 1st Generation1st Generation O H N P 2nd GenerationC 2nd GenerationP OH 3rdO GenerationP 3rd GenerationC P OH 1st Generation1st GenerationH2 N 2 2nd Generation2nd Generation 3rd Generation3rd Generation H2N H2N 1st GenerationOH CH3 O H N 2nd Generation 3rd Generation 2 H2N N 2 OH CH O OH CH3 O 3 H N N N OH CH3 OH O CH3 O 2 OH OH N OH N OH CH3 OH O CH3 O OH OH OH N N 7 N OPEtidronateC P OH N N OPOPC P C OHP OH OH O OHN N OH OH O 4 O OH OH N N OPOPC P C OH P OH O RisedronateO O OPOPC P C OH P OH OH OH O O OH OH OH CH3 O OH OH O O OH OH O O N Alendronate O O OH OHOH OHOH OH O P O C P P C OH P OH OH OH OH O OP O C PNHP P CC OH P P OH OH O P C P OH OH OH OH OH OH OH O P 2 O C P P C OH P OH O P O C P P C OH P OH OH OH OH OH OH OH O P O C P P C OH P OH O P O C P P C OH P OH 2 2 2 2 2 OH OH OH OH OH OH 2 2 7 7 N OH OH OH OH OH OH OH OH OHOHOH OHOH OHOH N OH OH OH OH OH OH OH OH OHOH OH7 OH 7 OPEtidronateC EtidronateP OH OHOH OHOH OH OH OHOH OH 7 7 7 EtidronateEtidronate EtidronateEtidronateEtidronate 4 4 OH RisedronateORisedronate OH OH 4 O 4 RisedronateRisedronate O AlendronateAlendronate4 4 4 RisedronateRisedronate AlendronateAlendronate OHRisedronate NH2 AlendronateNH2 Alendronate AlendronateNH2 NH2 NH2 NH2 N N O P NH2 C P OH OH OH OH O P N C NNH2 O P C P OH O P C P N OH N

OH OH O O N OH OH O O OH OH OH OH OH OH O O 8 2 OH OH OH OH OH OH OH O P O C P P C OH P OH O P C NH OH OH OH OH Cl O OH 5 O O P C NH2 2 O P O C P P C OH P OH O P OC NHP C NH O P O C P POHC OH P OH OH O POH CZoledronateNH2 2 7 O OHP 2 C NH2 Pamidronate OH OH OH OH 8 8 Etidronate OH OH OH OH OH OH OH OH OH OH OH OH OH8 OH 8 OH OH OH OH OH OH 8 8 OPC P OH O POH OHC OH OH POH OHOH O P C NH OH Cl OH O Cl O 5 5 4 2 OH Cl OH O Cl O 5 5 ZoledronateZoledronate OH Cl OH O Cl O 5 5 Risedronate PamidronateHPamidronate2N ZoledronateZoledronate ZoledronateZoledronate PamidronatePamidronate OH OH OPOPC P C OHP OH PamidronatePamidronateAlendronate 8 OH OH OH OH OPCl OPOHC P C OH P OH OPOPC P C OH P OH H N H2N OH Cl O 2 NH H2N H52N 2 3 H2N H2N Zoledronate OH Cl OH OHCl OH OH Cl OH OH Cl OH OH Cl OH OH Cl OH Pamidronate Clodronate3 3 3 3 N OPC P 3 OH 3 ClodronateClodronate N ClodronateClodronate ClodronateClodronate H2N OH O N N N N OH OHO O N N OH Cl OH OH OOH O OH OOHOH O O O P C P OH O P OC PP COH P OH OH O 3 OH OHO O OH O P CO PP OHC P OH OH OHO O O P CO PP OHC P OH OH OHO O 6 6 6 OH OH OHOHOH OH OHOH 6 O P 6 O C P P C OH P OH Clodronate OH OH OHOH OH6 OH 6 O P C P OH OH OH OH OH OH O P OC PP COH P OH O P OC PP COH P OH O P C P 9 OH9 N O P C NH OH OH OHOH OH OH 2 NeridronateNeridronate 9 9 9 OHNeridronate NeridronateO OH OHOH OHOH OHOH9 OH 9 NeridronateNeridronateNeridronate OH OH OHOH OH OH Ibandronate Ibandronate IbandronateIbandronate IbandronateIbandronateIbandronate O P C P OH OH OH OH O 8 OH OH OH 6 OH OH OH O P C P OH OH Cl O 5 Zoledronate 9 Neridronate OH OH OH Pamidronate

Ibandronate OPC P OH

H2N

OH Cl OH

3 Clodronate

OH O

O P C P OH OH O

6 OH OH OH O P C P OH

9 Neridronate OH OH OH

Ibandronate

Int. J. Mol. Sci. 2021, 22, 6869 3 of 21

In the treatment of osteoporosis, BPs are used, and they are active for some pathologies characterized by abnormally high bone resorption, which includes metastatic bone cancer, etc. [8,9]. BPs have experienced a huge development in the treatment of some bone diseases [10,11]. Currently, some of the uses of BPs are: X In corticosteroid and postmenopausal induced osteoporosis, the most utilized BPs in these cases, which block the presence of pathological fractures, is alendronate [12]. X They are also utilized to enhance bone morphology and decrease pain in Paget’s disease [13]. X In hypercalcaemia of malignancy, its role is in trying to check hypercalcaemia, reducing pain, and preventing the development of osteolitic lesions and fractures [14]. X In people with bone metastasis, it is utilized to decrease fractures, hypercalcemia, and relieve pain [15,16]. X They are used to decrease associated bone pathologies in multiple myeloma, including pain, fractures, and vertebral collapse [17,18]. Despite the efﬁcacy of BPs in the treatment of many bone-related diseases and their relatively good record, BPs also suffer from several limitations such as diarrhea, esophageal erosion, gastric ulcers, rare cases of esophageal cancer, hepatotoxicity, conjunctivitis and hypocalcemia, bone pain, fever, osteonecrosis of the jaw, and femur fracture [19–22]. This review focuses on bisphosphonate-based conjugate compounds developed for treating osteoporosis, bone cancer, and cancers that spread to the bones, such as prostate, lung, and breast cancer.

2. Osteoporosis Osteoporosis is a leading public health challenge, and it has become one of the most dominant chronic diseases in the world [23]. It is the most widely recognized skeletal disease that is initiated by an imbalance in bone metabolism, leading to a gradual loss of bone mass and attenuation of bone strength, thus disposing to low-energy fractures [24]. It is common in older adults, more especially postmenopausal women. This disease is characterized by bone loss that results in bone fractures in the spine and hip [25]. In developed countries such as the United States, the number of individuals with osteoporosis is increasing due to the increasingly aged population. Therefore, the treatment and prevention of osteoporosis are of great concern [25]. The estimates of women in danger of osteoporotic fractures and the respective economic burden to the health care systems in the next decades are expected to grow continuously [24]. In 2005, the cost for the treatment of fractures due to osteoporosis in the United States was $16.9 billion; it is predicted that the cost will increase to $25.3 billion by the year 2025 [23]. The pharmacological agents utilized in the management of osteoporosis include medications that precisely inhibit bone loss, decrease the risk of future fractures, and also increases bone strength [26]. In patients with osteoporosis, BPs are known drugs that inhibits the loss of bone mass. The therapeutic efﬁcacy of BPs is based on their remarkable selectivity to the bone rather than other tissues [27]. Many BPs have been approved and utilized to manage osteoporosis, e.g., alendronate, risedronate, zoledronate, ibandronate, etc., in several countries [26,28]. BPs decrease fracture risk by 30–70% at the spine and increase bone mass. Furthermore, they do the same at varying degrees at other skeletal sites in postmenopausal women [28,29]. Risk factors of osteoporosis include: X Age: the people at high risk are individuals who are at the age of 65 and above. X Gender: men are at less risk than women because of the absence of menopause [30]. X Family ancestry plays a signiﬁcant role. X Drinking too much alcohol and smoking are risk factors for osteoporosis [31].

Bisphosphonates Conjugates for the Treatment of Osteoporosis New BP-conjugate anti-osteoporotic agents have been designed and are presented in Figure2. Compounds 10, 11, 12, 13, 14 were not effective compared to the drugs that Int. J. Mol. Sci. 2021, 22, 6869 4 of 21

are already utilized to treat osteoporosis [32]. Therefore, there is still a need to develop drugs that will be effective as anti-resorptive agents for treating osteoporosis. It is reported that vitamin D conjugates for effective for improving good bone health; hence, Steinmeyer et al. synthesized a series of novel vitamin D conjugates by combining bisphosphonate moieties and vitamin D [33]. The in vitro evaluation of vitamin D conjugates (15, 16 and 18, 19) exhibited moderate activities, while 17 showed a highly moderate vitamin D-like activity in HL 60 and ROS 17/2.8 [33]. Tsushima et al. synthesized and evaluated the effects of a new hybrid compound of estrogen-bisphosphonate, 20 [34]. The results showed that 20 is an effective bone-targeting therapeutic, and it also displayed a strong ability to inhibit bone loss. Therefore, utilizing bisphosphonates for hybrid compounds is a promising approach for successful delivery of estrogen to the bone [34]. Xie et al. prepared and characterized a new bone-targeting conjugate, methotrexate-alendronate, 21. 1H- NMR, 13C-NMR, 31P-NMR, and LC-MS were used to characterize the conjugate [35]. The conjugate compound prevented bone resorption and the formation of osteoclast compared to methotrexate and alendronate utilized alone. Compound 21 potential to adhere to the exposed bone surface and improve drug accumulation in the pathological region for targeted therapy against osteoclastogenesis was signiﬁcant. Therefore, the results revealed methotrexate-alendronate conjugate as a potential therapeutic for the treatment of rheumatoid arthritis and osteoporosis by targeting osteoclastogenesis [35]. Motaleb et al. synthesized and characterized a novel zoledronate conjugate, 22, and successfully radiolabeled with 99mTc [36]. Compound 22 displayed excellent biological activities with high preferential uptake to the bone with clearance from blood, soft tissues; and these results suggest that zoledronate conjugates is a promising drug as an anti-osteoporotic Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 23 agent [36].

OH OH PO3H2 PO3H PO3H2 N PO3H2 N PO H 3 2 PO H X H2N N 3 2 12 N 11 10 X-F

PO3H2

OH HN

PO3H2 PO3H2 O PO3H2 O 14 13

O R MeO RO O H P OMe P O R: OR EtO P 15 OH H OEt 16

O O EtO OEt P P 17 EtO O OEt O EtO HO OH P OEt 18 P OEt P O EtO EtO OEt O 19

OH H NaHO3P PO HNa Me O 3 O

PO3HNa N N H HN PO3HNa N HO Me 20 O O HO

HN H NaHO3P PO3H2 OH 22 NH2 O

N N N 21 H

H2N N N Figure 2. Structures of BP‐conjugates 10–22, potential compounds reported for the treatment of Figureosteoporosis. 2. Structures of BP-conjugates 10–22, potential compounds reported for the treatment of

osteoporosis.3. Bone Cancer Bones are crucial in the body for movement and structure, mineral storage, protection of organs, and in blood cell production, provides a site as bone marrow houses hemato‐ poietic cells for the production of leukocytes, platelets, and red blood cells [37]. Bones are essential for storing phosphorus and calcium in the body because of their hydroxyapatite crystal structure. Bones continuously undergo cycling of resorption and deposition of the osteoid and hydroxyapatite matrix. Bone releases minerals via resorption by the osteo‐ clasts, the cells accountable for separating bone matrix, whereas osteoblasts develop novel bone through emitting chondroitin, osteocalcin, and collagen to formulate osteoid [37].

Int. J. Mol. Sci. 2021, 22, 6869 5 of 21

3. Bone Cancer Bones are crucial in the body for movement and structure, mineral storage, protection of organs, and in blood cell production, provides a site as bone marrow houses hematopoi- etic cells for the production of leukocytes, platelets, and red blood cells [37]. Bones are essential for storing phosphorus and calcium in the body because of their hydroxyapatite crystal structure. Bones continuously undergo cycling of resorption and deposition of the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW osteoid and hydroxyapatite matrix. Bone releases minerals via resorption6 of 23 by the osteo-

clasts, the cells accountable for separating bone matrix, whereas osteoblasts develop novel bone through emitting chondroitin, osteocalcin, and collagen to formulate osteoid [37]. The balance betweenThe balance bone between formation bone and formation resorption and relies resorption upon signals relies from upon chemokines, signals from chemokines, cytokines, and mechanostimulation. Moreover, resorption by osteoclasts is important cytokines, and mechanostimulation. Moreover, resorption by osteoclasts is important for for the maintenance of typical bone density. An imbalance in these procedures results the maintenance of typical bone density. An imbalance in these procedures results in hy‐ in hyperplastic bone, including mutual degenerative bone infections [37]. A schematic perplastic bone, including mutual degenerative bone infections [37]. A schematic illustra‐ illustration of bone resorption and formation is shown in Figure3. tion of bone resorption and formation is shown in Figure 3.

Figure 3.Figure A schematic 3. A schematic diagram diagram illustrating illustrating bone resorption bone resorption and bone and formation. bone formation.

For cancer patients,For cancer it is reported patients, that it is reportedcancer usually that cancer spreads usually to the spreads bone. Metastatic to the bone. Metastatic bone cancer happensbone cancer mostly happens in patients mostly with in patients prostate, with lung, prostate, and breast lung, cancer and breast [38]. cancer The [38]. The re- resulting skeletalsulting events skeletal include events spinal include cord spinal compression, cord compression, pathologic pathologic fractures, fractures, and bone and bone pain. pain. MetastasisMetastasis occurs when occurs cancer when cells cancer spread cells from spread the from primary the primary cancer cancersite to an site inac to an‐ inaccessible cessible site throughsite through the circulatory the circulatory system system forming forming secondary secondary cancer cancer [39]. The [39]. reported The reported median median survivalsurvival rate for rate people for people with withmetastatic metastatic bone boneresulting resulting from from breast, breast, renal, renal, and and prostate prostate cancercancer ranges ranges from from 12 to 12 33 to months 33 months [35]. [ The35]. Thesurvival survival rates rates for people for people with with pri‐ primary lung mary lung cancercancer are arevery very low low and andrange range from from 9.5% 9.5%to 12% to in 12% 12 months in 12 months [38]. People [38]. with People with bone bone metastasesmetastases can experience can experience some limitations some limitations in their in daily their lives, daily lives,such as such increased as increased medical medical expenses,expenses, decreased decreased quality quality of life, of and life, the and threat the threat of survival of survival [40]. [40]. OsteosarcomaOsteosarcoma is a well‐known is a diagnosed well-known primary diagnosed bone primary malignancy bone [41]. malignancy Osteosar [‐41]. Osteosar- coma generallycoma occurs generally either occurs during either active during growth active phases growth in phases adolescence in adolescence and young and young adults adults or oftenor associated often associated with a with pre‐existing a pre-existing condition, condition, like Paget’s like Paget’s disease disease (in adults (in adults above above 65) [41].65) The [41 occurrence]. The occurrence of bone cancer of bone in males cancer is inhigher males than is higherin females. than However, in females. However, osteosarcoma osteosarcomain females develops in females earlier develops compared earlier to males compared [42]. toMetastatic males [42 cancer]. Metastatic affects cancer affects the skeleton. Inthe particular, skeleton. cancers In particular, with a cancers high incidence with a high and incidence relatively and long relatively clinical course long clinical course include prostate,include lung, prostate, and breast lung, cancers, and breast and they cancers, often and metastasize they often to metastasize the bone [43–45]. to the bone [43–45]. Most primary cancers and bone metastases exhibit improved osteoclast action and bone resorption [46,47] that can lead to hypercalcemia, pathological fractures, and pain [45]. Therefore, techniques to decrease the incidence and morbidity of bone metastases are ob‐ viously of tremendous clinical significance [41]. The treatment that is currently used is the combination of chemotherapeutics and surgery. Thecombination of high dosages of meth‐ otrexate, cisplatin, ifosfamide, and doxorubicin have prompted an increased survival rate. Nonetheless, the utilization of anticancer medications is limited by severe side effects. The poor bone blood supply, drug‐resistance, and non‐specific uptake require the utilization of high toxic doses of anticancer drugs [41]. Hence, there is a need to develop bone‐

Int. J. Mol. Sci. 2021, 22, 6869 6 of 21

Most primary cancers and bone metastases exhibit improved osteoclast action and bone resorption [46,47] that can lead to hypercalcemia, pathological fractures, and pain [45]. Therefore, techniques to decrease the incidence and morbidity of bone metastases are obviously of tremendous clinical signiﬁcance [41]. The treatment that is currently used is the combination of chemotherapeutics and surgery. Thecombination of high dosages of methotrexate, cisplatin, ifosfamide, and doxorubicin have prompted an increased survival rate. Nonetheless, the utilization of anticancer medications is limited by severe side effects. The poor bone blood supply, drug-resistance, and non-speciﬁc uptake require the utilization of high toxic doses of anticancer drugs [41]. Hence, there is a need to develop bone-targeted anticancer agents with reduced and/or no side effects for the treatment and prevention of malignant growth-related bone diseases [48]. Numerous therapeutic techniques have been developed for the management of malignant- related skeletal complications. These approaches incorporate either anti-neoplastic interven- tion, for example, chemotherapy, hormonal therapies, radiotherapy, and topical surgeries, or bone helpful treatment such as calcium, analgesics, and vitamin D supplements [39]. In the treatment of malignant bone metastasis, synthetic anti-resorptive agents are considered to be indispensable [39]. BPs are known as the most effective of these drugs. They have an established role as palliative treatment in people with skeletal metastases. BPs increases the prevention of cancer cell invasion and adhesion to the bone matrix and induces apoptosis of osteoclasts [39]. Some studies reported that BPs stimulate osteoblastic bone formation in vitro and in vivo. BPs is one class of pharmacological agents used for the prevention and management of myeloma bone disease. However, agents like anti-RANKL antibody, Deno- sumab are also utilized in people with bone metastasis resulting from prostate and breast cancer. Denosumab is currently under clinical trials in patients with Multiple myeloma [49]. Anticancer agents, for example, doxorubicin, cisplatin, and methotrexate were, covalently incorporated into the terminal amino group of the amino-BP alendronate utilizing a suit- ably bio-reversible amide bond. The BP-conjugates displayed prolonged blood circulation, binds to the hydroxyapatite matrix of the bone, and hence released the BP together with the cytostatic agent into the bone micro-environment via the cleavage of the amide linker of the BP-conjugate [50].

BP Conjugates as Bone-Targeting Compounds The administration of BPs utilized in clinical practice is usually via intravenous or oral routes. Oral administration of BPs is challenging due to poor gastrointestinal tolerability and bioavailability [32]. Due to their high afﬁnity to the bone, between 30% and 60% of the absorbed drug quickly binds to bone mineral. This feature is utilized to build drug BP-conjugates that can be considered a good approach for selective drug targeting to the bone. Such an effective transport of therapeutic agents to the bone is called osteotropic drug delivery system (ODDS). It decreases drug toxicity and enhances its bioavailability at the desired site. The effective transportation of bioactive agents to the bone is named osteotropic drug delivery system [32]. The development of bone metastases is one key reason for tumor-related death. Hence, it is important to design anti-tumor drugs that target bone tissue for enhanced therapeutic outcomes. Polymer-based carriers incorporated with BPs together with anti-tumor agents have been developed for synergistic anticancer effects. Bisphosphonate was conjugated to camptothecin for bone targeting [51]. Additionally, the combination of BPs with anti-cancer drugs, which include bortezomib, doxorubicin, gemcitabine, or camptothecin, has been effective against multiple myeloma cells. Examples of BP-conjugates containing anti-cancer drugs are shown in Figure4[ 51,52]. Agyin et al. prepared a sequence of proteasome inhibitor BP-conjugates that are effective inhibitors of proliferation and cell viability in vitro in myeloma cell lines. Table2 shows the in vitro antiproliferative and cytotoxic effect of the bone-targeted proteasome inhibitors of compounds 24, 25, and 26. The IC50 values of compounds 24 and 25 were moderate, while the IC50 value of compound 26 showed synergistic effects on 5TGM1 cell lines [52]. According Int. J. Mol. Sci. 2021, 22, 6869 7 of 21

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 23 to the results, the compounds are potential bone-targeted agents for the treatment of multiple myeloma.

O N O H N N N B(OH)2 R: N H O 24 23 HN O PO H 3 2 N HO H O N R O O O H2O3P O O H2O3P NH O H N N B(OH)2 H2O3P N 25 H O N

O O H N O N N B(OH)2 26 H H O

Figure 4. The structures of BP BP-conjugates‐conjugates 23–26, compounds with good bone-targetingbone‐targeting capability.

Table 2. Anti-proliferativeTo develop and an cytotoxic active effects agent offor the the bone-targeted treatment of proteasome bone metastases, inhibitors. some BP‐conjugates with platinum(II) complexes, such as cisplatin (since it has a broad spectrum range of can‐ cer drugs), have been synthesizedIC50 (nM) and evaluated for the treatment of bone metastases [53]. Cell Line PS-341Nakatake et PS-341-BP-1 al. synthesized (24) cis‐ PS-341-BP-2diammine (P,P (25)‐diethyl MG-262methylenebisphosphonato) MG-262 BP (26) plati‐ 5TGM1 6.78 +num(II) 0.57 (DEBP‐ 7.59Pt) +and 0.68 intensively 9.39evaluated + 0.78 the impact 9.18 on + 0.54the cancer cells 4.89 and + 0.12 bone re‐ sorption activity of osteoclast Figure 5. Cell development inhibitory impact of DEBP‐Pt RPMI 8226 9.50 + 0.48 10.18 + 0.87 11.51 + 0.92 13.76 + 0.89 12.47 + 0.87 (21) was examined against four cancer cell lines. The results in Table 3 revealed that com‐ pound 29 displayed anticancer activity, and the impact of compound 29 on bone resorp‐ tion activityTo develop was an also active evaluated. agent for Compound the treatment 29 demonstrated of bone metastases, a prominent some BP-conjugatesinhibitory im‐ withpact on platinum(II) bone resorption, complexes, as displayed such as cisplatin in Table (since 4. The it has results a broad indicated spectrum the range anticancer of cancer ac‐ tivitydrugs), and have bone been resorption synthesized inhibitory and evaluated effect of the for thecompound. treatment Therefore, of bone metastasesthe outcome [53 of]. theNakatake results etreveals al. synthesized the potential cis-diammine of compound (P,P-diethyl 29 for the treatment methylenebisphosphonato) of metastatic bone plat-can‐ cerinum(II) [53]. (DEBP-Pt) and intensively evaluated the impact on the cancer cells and bone resorption activity of osteoclast Figure5. Cell development inhibitory impact of DEBP-Pt (21)Table was 3. Cytotoxicity examined againstof the DEBP four‐Pt, cancer compound cell lines. 29 against The results Various in Human Table3 Tumor revealed Cell that Lines. compound 29 displayed anticancer activity, and the impact of compound 29 on bone resorption activity was alsoCell evaluated. Lines Compound 29 demonstrated a prominentIC50 (mol/L) inhibitory impact on Int. J. Mol. Sci. 2021, 22, x FOR PEER boneREVIEW resorption, asHLC displayed‐2 in Table4. The results indicated2.6 the × anticancer10−5 activity and 9 of 23 bone resorptionHCC1954 inhibitory effect of the compound. Therefore,2.8 the × 10 outcome–5 of the results reveals the potentialMCF of‐7 compound 29 for the treatment of metastatic2.6 × 10–5 bone cancer [53]. K562 4.7 × 10–6 O OEt Table 4. Effect on Bone Resorption Activity of Osteoclast. O O O O P EtO OEt EtO OEt H3N O P P Substance P P ICPt50 (mol/L) EtO OEt HO OH H3N O –−6 DEBP‐Pt 2.8 × 10 P 27 Cisplatin 28 1.1 × 10–5 OEt 29 O FigureFigure 5. 5.BP-conjugate BP‐conjugate29. 29.

Ebetino et al. synthesized BP‐conjugates as shown in Figure 6. The phenyl conjugate NE‐58022 (30), NE‐58086 (31) of risedronate has a coupling mode almost the same as that of risedronate. However, it was less effective with IC50 values of 1626, 2588 nM (IC50 5.7 nM for risedronate), respectively. NE‐10575 (32) was an effective conjugate with an IC50 value of 19.6 nM. According to the results, compound 32 is a potential bone antitumor drug [54].

PO3H2 PO3H2 OH OH N PO3H2 PO3H PO3H2 N 30 31 32 H2O3P Figure 6. The structures of BP‐conjugates 30–32, risedronate containing compounds.

Kunda et al. synthesized new BP‐conjugates (33–42) (Figure 7) using NaH/PEG with a good percentage yield ranged between 59% and 71% [55]. All the conjugates were puri‐ fied by column chromatography and characterized by IR, 1H‐, 31P‐NMR, and MS spectros‐ copy.

N N HN R:

O N O O O 33 34 H 35 EtO OEt P P HO N EtO OEt N N

N NH2 36 CN 39 37 38

O N 2 40 41 H N NO 2 42 2 NH2 Figure 7. The structures of BP‐conjugates 33–42 reported by Kunda et al. [55].

In another study, it was proposed that the DTPA/BP chelate would allow medicinal radionuclides to target the bone. In contrast, the 5‐fluorouracil BP‐conjugate might be spe‐ cifically transported to metastases bone without systemic toxicity. El‐Mabhouh et al. pre‐ pared 188Re‐5‐FU/BP (43) and 188Re‐DTPA/BP (44) with good stability and high radiochem‐ ical yields, as shown in Figure 8 [56]. The compounds’ high bone uptake, particularly in the joints, low soft‐tissue uptake, and rapid plasma clearance, was significant. This in‐ creases the potential of specific treatment to metastatic bone lesions while avoiding com‐ plete toxicity. Therefore, BP‐conjugates are promising therapeutics for the management of bone metastases [56].

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Table 3. Cytotoxicity of the DEBP-Pt, compound 29 against Various Human Tumor Cell Lines. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 23 Cell Lines IC50 (mol/L) Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 23 HLC-2 2.6 × 10−5 HCC1954 2.8 × 10−5 O −5 MCF-7 OEt2.6 × 10 O O O O O P −6 K562 OEt4.7 × 10 EtO OEt EtO OEt H3N O P OP OP OP OP Pt H N O EtO OEt EtOHO OEtOH H33N O P P P P Pt P Table 4. Effect27 on Bone Resorption28 Activity of Osteoclast. EtO OEt HO OH H3N O OEt 29 P 27 O 28 OEt 29 SubstanceO IC50 (mol/L) Figure 5. BP‐conjugate 29. DEBP-Pt 2.8 × 10−6 Figure 5. BP‐conjugate 29. Cisplatin 1.1 × 10−5 Ebetino et al. synthesized BP‐conjugates as shown in Figure 6. The phenyl conjugate NE‐58022Ebetino (30), et NEal. synthesized‐58086 (31) of BP risedronate‐conjugates has as showna coupling in Figure mode 6. almost The phenyl the same conjugate as that NEof risedronate.‐58022Ebetino (30), et al.NEHowever, synthesized‐58086 (31)it was of BP-conjugates risedronateless effective has as with showna coupling IC50 in values Figure mode of6 . almost1626, The phenyl 2588 the samenM conjugate (IC as50 that 5.7 NE-58022ofnM risedronate. for risedronate), (30), NE-58086 However, respectively. (31) it was of risedronate less NE effective‐10575 has (32) with a coupling was IC 50an values modeeffective of almost 1626, conjugate the 2588 same nMwith as (IC thatan50 IC 5.7 of50 risedronate.nMvalue for of risedronate), 19.6 However, nM. According respectively. it was less to the effective NE results,‐10575 with compound(32) IC was50 values an 32 effective ofis 1626,a potential conjugate 2588 nM bone (ICwith antitumor50 5.7an nMIC50 forvaluedrug risedronate), [54].of 19.6 nM. respectively. According NE-10575to the results, (32) was compound an effective 32 is conjugate a potential with bone an ICantitumor50 value ofdrug 19.6 [54]. nM. According to the results, compound 32 is a potential bone antitumor drug [54].

PO3H2 PO3H2 OH PO H PO H OH3 2 PO3H2 N 3 2 PO H OH 3 PO3H2 PO H N OH 30 N 31 3 2 32 PO H H O P 3 PO3H2 2 3 N 30 31 32 H O P Figure 6. The structures of BP‐conjugates2 3 30–32, risedronate containing compounds. FigureFigure 6.6. TheThe structuresstructures ofof BP-conjugatesBP‐conjugates 30–32,30–32, risedronaterisedronate containingcontaining compounds. compounds. Kunda et al. synthesized new BP‐conjugates (33–42) (Figure 7) using NaH/PEG with a goodKundaKunda percentage etet al. al. synthesized synthesized yield ranged new new between BP-conjugates BP‐conjugates 59% and (33–42) (33–42) 71% [55]. (Figure (Figure All7 the) using7) conjugatesusing NaH/PEG NaH/PEG were with puriwith a ‐ goodafied good by percentage percentagecolumn chromatography yield yield ranged ranged between between and characterized 59% 59% and and 71% 71% by [55 IR, ].[55]. All 1H All‐ the, 31 Pthe conjugates‐NMR, conjugates and were MS were spectros puriﬁed puri‐ 1 31 byfiedcopy. column by column chromatography chromatography and characterized and characterized by IR, byH-, IR, 1HP-NMR,‐, 31P‐NMR, and and MS spectroscopy.MS spectros‐ copy. N N HN R: N N HN R: O N O O O 33 34 H 35 EtO OEt O N O O O 33 34 P P H 35 EtO OEt HO N EtO OEt N N P P HO N EtO R OEt N N N NH R 2 36 CN 39 37 38 N NH2 36 CN 39 37 38

O N 2 40 41 H2N NO2 O2N 42 40 NH2 41 H N NO 2 42 2 NH2 FigureFigure 7.7. TheThe structuresstructures ofof BP-conjugatesBP‐conjugates33–42 33–42reported reported by by Kunda Kunda et et al. al. [ [55].55]. Figure 7. The structures of BP‐conjugates 33–42 reported by Kunda et al. [55]. InIn anotheranother study,study, it it was was proposed proposed that that the the DTPA/BP DTPA/BP chelatechelate would would allow allow medicinal medicinal radionuclidesradionuclidesIn another to study, target target itthe the was bone. bone. proposed In In contrast, contrast, that thethe the DTPA/BP5‐fluorouracil 5-fluorouracil chelate BP‐ BP-conjugateconjugatewould allow might medicinal might be spe be‐ specificallyradionuclidescifically transported transported to target to themetastases to bone. metastases In bonecontrast, bone without withoutthe 5 systemic‐fluorouracil systemic toxicity. BP toxicity.‐conjugate El‐Mabhouh El-Mabhouh might et beal. et spepre al.‐ preparedcificallypared 188 transportedRe188‐5Re-5-FU/BP‐FU/BP (43)to metastases (43)and and188Re188‐ boneDTPA/BPRe-DTPA/BP without (44) systemic with (44) good with toxicity. good stability stability El and‐Mabhouh high and radiochem high et al. radio- pre‐ chemicalparedical yields, 188Re yields, ‐as5‐ FU/BPshown as shown (43) in Figure and in Figure 188 Re8 [56].‐8DTPA/BP[ 56 The]. The compounds’ (44) compounds’ with good high high stability bone bone uptake, and uptake, high particularly particularly radiochem in‐ inicalthe the joints,yields, joints, lowas low shown soft soft-tissue‐tissue in Figure uptake, uptake, 8 [56]. and and The rapid rapid compounds’ plasma plasma clearance, high clearance, bone was uptake, was significant. significant. particularly This This inin‐ increasesthecreases joints, the the low potential potential soft‐tissue of ofspecific specificuptake, treatment treatmentand rapid to metastatic toplasma metastatic clearance, bone bone lesions was lesions while significant. while avoiding avoiding This com in‐ completecreasesplete toxicity. the toxicity. potential Therefore, Therefore, of specific BP BP-conjugates‐conjugates treatment are to are promisingmetastatic promising bonetherapeutics therapeutics lesions whilefor for the avoiding managementmanagement com‐ ofpleteof bonebone toxicity. metastasesmetastases Therefore, [[56].56]. BP‐conjugates are promising therapeutics for the management of bone metastases [56].

Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, 6869x FOR PEER REVIEW 109 of 2123

OH H H2O3P N N OH H2O3P N N OH N N

PO3H2 O OH HO O PO3H2 O OH HO O

O O O 43 O

H O N O H2O3P CH3 H2O3P CH3 H H2O3P N N H O P OH H2O3P N N H2O3P OH X 2 3 X 45 44 PO3H2 O 44 PO3H2 O X-F X-F FigureFigure 8.8. TheThe structuresstructures ofof BP-conjugatesBP‐conjugates 43–4543–45 reportedreported byby El-MabhouhEl‐Mabhouh et et al. al. [ [56].56].

Tumor-inducedTumor‐induced bonebone diseasedisease (TIBD)(TIBD) isis oneone ofof thethe signiﬁcantsignificant reasonsreasons forfor mortalitymortality andand morbiditymorbidity inin peoplepeople withwith malignantmalignant growth.growth. TheThe developmentdevelopment ofof anan activeactive drugdrug forfor thethe treatmenttreatment ofof TIBDTIBD isis necessarynecessary becausebecause itit causescauses extensive extensive morbiditymorbidity [52[52].]. TIBDTIBD isis commoncommon inin cancercancer patientspatients and and a a medianmedian survival survival of of less less thanthan twotwo years.years. TheThe developmentdevelopment ofof BP-conjugatesBP‐conjugates with with improved improved anti-resorptive anti‐resorptive and and cytotoxic cytotoxic is a is pressing a pressing need need to enhance to en‐ thehance medication the medication for patients for patients with TIBD. with BP-conjugatesTIBD. BP‐conjugates can be can utilized be utilized effectively effectively as bone- as targetingbone‐targeting therapeutics. therapeutics. In addition, In addition, BP-conjugates BP‐conjugates can enhancecan enhance the controlthe control of disease- of dis‐ associatedease‐associated symptoms, symptoms, reduce reduce the intrusion the intrusion of remedy of remedy on the on events the events of everyday of everyday life, and life, reduceand reduce treatment-related treatment‐related toxicity toxicity [57]. BP-conjugates, [57]. BP‐conjugates, (MBC-1 (MBC (46),‐ MBC-111 (46), MBC (47),‐11 MBC-9 (47), (48),MBC and‐9 (48), MBC-29 and MBC (49))‐ as29 shown (49)) as in shown Figure 9in. TheFigure results 9. The showed results that showed compound that compound 47 can be delivered47 can be todelivered the bone to and the itbone displayed and it displayed anti-tumor anti activity‐tumor with activity prolonged with prolonged survival rate, sur‐ makingvival rate, it a making promising it a therapypromising for therapy TIBD [57 for]. TIBD [57].

O CH3 O O O CH3 O O

HO P C P O P OR

OH OH OH OH

O NH2 NH2 O NH2 O NH2 F NH F N F NH N N O N O N O N O O N O N O R: O O O 46 47 49 OH 48 OH 48 OH OH HO OH OH HO HO HO FigureFigure 9.9. BP-conjugatesBP‐conjugates46–49, 46–49,potential potential compounds compounds for for the the treatment treatment of Tumor-inducedof Tumor‐induced bone bone disease. disease. 4. Metastatic Cancer That Spreads to the Bones 4. MetastaticBone metastasis Cancer that is linked Spreads with to most the Bones types of cancers leading to high mortality and morbidityBone metastasis in cancer patients. is linked However, with most it types is most of cancers prevalent leading in people to high living mortality with lung, and breast,morbidity and in prostate cancer cancerpatients. [58 However,,59]. Although it is most bone prevalent metastasis in ispeople common living in with myeloma, lung, prostate,breast, and and prostate breast cancer, cancer according [58,59]. Although to radiographic bone metastasis appearance, is the common primary in pathogene- myeloma, sisprostate, often includes and breast both cancer, osteolytic according and osteoblasticto radiographic processes appearance, [60]. About the primary 75% of pathogen patients‐ sufferesis often from includes severe pain both during osteolytic the time and of osteoblastic diagnosis of processes metastatic [60]. bone. About It may 75% lead of topatients SREs, suchsuffer as from hypercalcemia, severe pain pathological during the time fractures, of diagnosis and spinal of metastatic cord compression bone. It resultingmay lead into increasedSREs, such medical as hypercalcemia, care cost, shortened pathological survival fractures, rate, and and decrease spinal cord quality compression of life [59]. result Some‐ researchersing in increased reported medical that BPs care could cost, prevent shortened tumor survival cell invasion, rate, and angiogenesis, decrease quality proliferation of life and enhance survival rate in vivo [61]. BPs have also been utilized to minimize pain in [59]. Some researchers reported that BPs could prevent tumor cell invasion, angiogenesis,

Int. J. Mol. Sci. 2021, 22, 6869 10 of 21

multiple myeloma and reduce skeletal complications [62]. In addition, BPs can slow down bone-resorption and exert cytotoxic effects on osteoclasts [63]. In people with cancer, BPs can affect the bone micro-environment to decrease the invasion of the cancer cells [64]. BPs also exhibit anti-angiogenic and anti-tumor activity [65–70]. However, the biological diversity of bone metastasis and tumors can result in different responses to BP remedy. Moreover, BP drug can expose patients to serious side effects such as osteonecrosis of the jaw, hypocalcemia, and nephrotoxicity [71]. Osteonecrosis of the jaw is one of the most severe side effects. Most cases of bisphosphonate- related osteonecrosis of the jaw have been reported in people with breast cancer and multiple myeloma treated with high doses of intravenous bisphosphonates. Moreover, it has also been reported in people taking bisphosphonates for the treatment of osteoporosis. The risk factors for bisphosphonate-related osteonecrosis of the jaw comprise the usage of higher dose, intravenous bisphosphonates and prolonged duration of administration. Furthermore, the usage of anticancer therapy and glucocorticoids, smoking, cancer, and history of diabetes are additional risk factors [37,72]. Hypocalcemia is also a common problem associated with bisphosphonate usage; moreover, the incidence can be as high as 18%. Hypocalcemia is more common, secondary to intravenous bisphosphonates, and in people with hypoparathyroidism, an underlying untreated vitamin-D deficiency, poor calcium intake, and hypocalcemia. Vitamin D and calcium deficiency should be treated before administering bisphosphonates, especially intravenous bisphosphonates [73,74]. Some BPs such as pamidronate, zoledronate, clodronate, and ibandronate have been in clinical use for the management of health problems associated with tumors that cause osteolysis. However, there is an increasing evidence from preclinical studies that demonstrate BPs good antitumor activity [75,76]. Numerous investigations have revealed a decrease in the risk of colorectal and breast cancer among BP users. Therefore, BPs can be clinically effective for the prevention and treatment of tumors [77].

4.1. Breast Cancer Globally, breast cancer is the leading cause of death amongst women. The World Health Organisation (WHO) reported that over 1.38 million women suffered from breast cancer in 2008 [78]. In 2016, roughly 61,000 cases of non-invasive breast cancer and 246,660 cases of invasive breast cancer were reported by the American Cancer Society (ACS), with 40,730 deaths [78]. It is estimated that between 65% and 75% of patients with breast cancer may suffer from cancer spreading to the bones with problems, such as hypercalcemia, spinal cord compression, and pathological fractures [79]. Strategies to preserve bone health are therefore an important aspect of breast cancer treatment [80]. Treatment approaches for the management of breast cancer are limited. Chemotherapeutic drugs are inhibitors of many metabolic pathways, but there are severe side effects such as fatigue, nausea, loss of appetite, hair loss, etc. [78]. Several studies proved N-BPs to be active therapies for the treatment of bone metastases in breast cancer patients. The oral use of BPs is associated with a reduced risk of colorectal and breast cancers [81]. Furthermore, BPs inhibit SRE and cancer therapy-induced bone loss in bone metastatic breast cancer [82]. Studies in preclinical trials reported the anticancer effect of BPs. Some epidemiologic research observed a beneficial effect of using BP in inhibiting breast cancer. Hence, numerous clinical trials studied the adjuvant usage of BP in early breast cancer [82]. For the prevention of SREs in postmenopausal women, zoledronate has been confirmed as a suitable bioactive agent. The largest group of patients with breast cancer are postmenopausal women with early-stage breast cancer, under estrogen- depleting treatment [83]. The study of breast cancer in Northern Israel revealed that the utilization of BPs for more than one year was linked with a 28% reduction in the risk of postmenopausal breast cancer, and these observations were confirmed by the Women’s Health Initiative (WHI) for oral BPs. Therefore, BPs reduce the risk of bone metastasis in high-risk breast cancer patients [83]. Pamidronate application in breast cancer has been demonstrated to decrease morbidity by decreasing the progression of bone metastases, reducing the need for radiation and pathological fractures. Similarly, clodronate has been Int. J. Mol. Sci. 2021, 22, 6869 11 of 21

reported to decrease the growth of new bone metastases in breast cancer patients, as well as the number of skeletal events [84].

4.1.1. In Vitro Studies of BPs in Breast Cancer Alendronate is one of the BPs utilized to reduce the risk of breast cancer in postmenopausal women. Ilyas et al. studied the cytotoxic effect of alendronate on HTB-132 malignant growth cell line. Alendronate was tested at 5, 10, 15, and 20 M, and the results showed a 47% cell death at 5 M, indicating that the treatment and dosage were most active at low concentration [78]. Additionally, alendronate can induce cytotoxic effects in HTB-132 cell lines at low concentrations and can be utilized as chemotherapeutic agents in breast cancer [78]. Body et al. determined the effects of N-BPs on the proliferation of malignant growth cell lines. The IC50 value of four N-BPs, which include alendronate, ibandronate, risedronate, and zoledronate, were observed in numerous cancer cell lines. Human cancer cell lines such as the GBM cell line U87, MDA-MD-43, and GBM patient-derived primary cell line SK429 were used. The results showed that zoledronate was effective with the lowest IC50 values ranging from 3 µM to 37 µM against all the cancer cell lines. Zoledronate exhibits an effective inhibitory effects on these different human cell lines [85].

4.1.2. Bisphosphonates Conjugates for the Treatment of Breast Cancer Schott et al. studied the therapeutic efﬁcacy of 5-Fluoro-20-deoxyuridine-alendronate (50) in breast cancer (Figure 10). Novel antimetabolite-bisphosphonates conjugates (5- 0 Fluoro-2 -deoxyuridine-alendronate) were prepared for bone targeting [50]. The IC50 and 0 IC90 values of 5-Fluoro-2 -deoxyuridine, 5-ﬂuorouracil, zoledronate, alendronate, and 5- Fluoro-20-deoxyuridine-alendronate on the cell line tested are shown in Table5. Compound 42 showed inhibitory effects in MCF-7 cell line with an IC50 value of 44.0 µM compared 0 to alendronate and 5-Fluoro-2 -deoxyuridine with an IC50 value of 55.9 µM and 51.3 µM, respectively. Compound 50 did not exhibit any inhibitory effects in the tested cell lines Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 0 13 of 23 compared with 5-Fluoro-2 -deoxyuridine, zoledronate, and alendronate [86]. In conclusion, the study indicates that compound 50 is toxic to osteoclasts in vitro but has an osteoblast and tumor cell-sparing effect compared to alendronate [87].

H O P 2 3 NH2 NH X-F HO PO3H2 X N N O CH3 O O

NaO P C P O P O HO N O O N O O OH OH OH OH 51 50 H OH OH OH H Figure 10.Figure Structures 10. Structures of BP of BP-conjugates‐conjugates with with anti-breast anti cancer‐breast activity cancer50, 51. activity 50, 51.

4.2. Prostate Cancer Bone metastases are the primary reason for morbidity in people with cancers like prostate, lung, breast, and multiple myeloma cancer. Specifically, breast and prostate can‐ cer target the skeleton as the preferred site of dissemination, representing 80% of all bone metastasis [88]. The second most dominant cancer globally is prostate cancer, with more than half a million men worldwide being diagnosed with prostate cancer [89,90]. US Can‐ cer Statistics stated that in 2014, 172,258 new cases were diagnosed, and 28,343 deaths were ascribed to prostate cancer disease in the country. Regardless of the attempt to treat the disease, about 6% of people with prostate cancer will grow skeletal metastasis during disease progression [90,91]. The clinical complications include fractures, pain, hypercalce‐ mia of malignancy, and compression of the spinal cord. The possibility of extensive sur‐ vival radically reduces when metastases occur since present treatments are relatively in‐ effectual [88,92,93]. The purpose of treating bone metastasis is to reduce and/or inhibit the complications of SREs, thus improving patients’ quality of life [94]. Though bone metas‐ tases in prostate cancer patients are common of the bone‐forming osteoblastic type, histo‐ logical and biochemical analyses proposed that there is also an addition of osteoclast ac‐ tion in these lesions, prompting an increased risk of SREs and bone destruction [87]. Os‐ teoclast inhibition restrains bone metastases in preclinical trials of prostate cancer. Conse‐ quently, osteoclast initiation plays a significant role in metastases development [95,96]. Bone metastasis causes negative results in patients with prostate cancer, and 80% of those with advanced prostate cancer have bone metastasis. Radiotherapy is the ideal ther‐ apy for bone metastasis. The complications of radiotherapy include pneumonia, myelo‐ suppression, and pancytopenia [97]. BPs are useful to inhibit fractures, skeletal complica‐ tions, and bone pain [96]. Some studies showed that osteolysis might be present in bone metastases prostate cancer [98]. BP treatment inhibits possible pathological fractures in men with prostate cancer metastases and reduces pain [97]. Zoledronate is the most potent BP, and it prevents negative results related to the bone in patients with prostate cancer and is linked to secondary bone metastasis [97]. Zoledronate and pamidronate are BPs that are accepted by the US FDA for the treatment and the prevention of the complications of SREs in prostate cancer metastatic patients [94].

4.2.1. In Vitro Studies of BPs in Prostate Cancer Zoledronate displays effective action of anti‐bone resorption and anti‐cancer activity [99]. Some in vitro preclinical studies demonstrated that zoledronate prevent tumor cell adhesion, thus damaging the progression of tumor cell metastasis and invasion. Further‐ more, it was reported that zoledronate has a significant effect in vitro stimulation on the angiogenesis of 𝛾/𝛿 T lymphocytes [100–102]. The tumor cell apoptosis is one of the crucial anticancer mechanisms of zoledronate [103]. In vitro and in vivo studies revealed a syn‐ ergistic anti‐tumor action of zoledronate when utilized in combination with either tar‐ geted molecular agents or cytotoxic drugs [103]. Pamidronate or zoledronate together with farnesyl transferase inhibitor (FTI) R115777 were utilized to assess the effects of the combination therapy on apoptosis and inhibition growth [104]. A synergistic effect was found between zoledronate and R115777 on both androgen‐dependent LNCaP and

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Table 5. In vitro evaluation of breast cancer cell line.

MCF-7 MDA-MB 231 OvCa-3 OvCa-29 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 5-ﬂuorouracil 25.5 44.2 24.4 52.1 1.2 6.3 10.7 30.0 Zoledronate 8.9 42.9 6.4 39.1 1.6 6.0 3.0 7.2 5-Fluoro-20-deoxyuridine- 51.3 83.8 12.8 62.5 24.5 64.7 8.2 62.5 alendronate Alendronate 55.9 100.7 32.3 69.7 12.7 39.8 16.6 38.2 5-Fluoro-20-deoxyuridine- 45.0 73.3 44.9 79.6 59.9 123.0 40.1 69.5 alendronate-aledronate

4.2. Prostate Cancer Bone metastases are the primary reason for morbidity in people with cancers like prostate, lung, breast, and multiple myeloma cancer. Speciﬁcally, breast and prostate cancer target the skeleton as the preferred site of dissemination, representing 80% of all bone metastasis [88]. The second most dominant cancer globally is prostate cancer, with more than half a million men worldwide being diagnosed with prostate cancer [89,90]. US Cancer Statistics stated that in 2014, 172,258 new cases were diagnosed, and 28,343 deaths were ascribed to prostate cancer disease in the country. Regardless of the attempt to treat the disease, about 6% of people with prostate cancer will grow skeletal metastasis during disease progression [90,91]. The clinical complications include fractures, pain, hypercalcemia of malignancy, and compression of the spinal cord. The possibility of extensive survival radically reduces when metastases occur since present treatments are relatively ineffectual [88,92,93]. The purpose of treating bone metastasis is to reduce and/or inhibit the complications of SREs, thus improving patients’ quality of life [94]. Though bone metastases in prostate cancer patients are common of the bone-forming osteoblastic type, histological and biochemical analyses proposed that there is also an addition of osteoclast action in these lesions, prompting an increased risk of SREs and bone destruction [87]. Osteoclast inhibition restrains bone metastases in preclinical trials of prostate cancer. Consequently, osteoclast initiation plays a signiﬁcant role in metastases development [95,96]. Bone metastasis causes negative results in patients with prostate cancer, and 80% of those with advanced prostate cancer have bone metastasis. Radiotherapy is the ideal therapy for bone metastasis. The complications of radiotherapy include pneumonia, myelo-suppression, and pancytopenia [97]. BPs are useful to inhibit fractures, skeletal complications, and bone pain [96]. Some studies showed that osteolysis might be present in bone metastases prostate cancer [98]. BP treatment inhibits possible pathological fractures in men with prostate cancer metastases and reduces pain [97]. Zoledronate is the most potent BP, and it prevents negative results related to the bone in patients with prostate cancer and is linked to secondary bone metastasis [97]. Zoledronate and pamidronate are BPs that are accepted by the US FDA for the treatment and the prevention of the complications of SREs in prostate cancer metastatic patients [94].

4.2.1. In Vitro Studies of BPs in Prostate Cancer Zoledronate displays effective action of anti-bone resorption and anti-cancer activity [99]. Some in vitro preclinical studies demonstrated that zoledronate prevent tumor cell adhesion, thus damaging the progression of tumor cell metastasis and invasion. Fur- thermore, it was reported that zoledronate has a signiﬁcant effect in vitro stimulation on the angiogenesis of γ/δ T lymphocytes [100–102]. The tumor cell apoptosis is one of the crucial anticancer mechanisms of zoledronate [103]. In vitro and in vivo studies revealed a synergistic anti-tumor action of zoledronate when utilized in combination with either targeted molecular agents or cytotoxic drugs [103]. Pamidronate or zoledronate together Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 23

Int. J. Mol. Sci. 2021, 22, 6869 13 of 21 androgen‐independent PC3 prostate cancer cell lines, and special effects were due to im‐ prove inactivation and apoptosis of Erk and Akt [104]. Moreover, De Rosa et al. reviewed with farnesyl transferase inhibitor (FTI) R115777 were utilized to assess the effects of thethe combination efficacy of therapy a combination on apoptosis therapy and inhibition of docetaxel growth [ 104and]. Azoledronate synergistic effect on hormone sensitive wasprostate found betweencancer zoledronatecell line, LNCaP. and R115777 A combination on both androgen-dependent of DTX and LNCaP ZOL andin hormone and drug androgen-independentrefractory, PC‐3 and PC3 DU prostate‐145 prostate cancer cell cancer lines, and cells special resulted effects in were a significant due to im- synergistic effect proveresulting inactivation from andinhibited apoptosis cell of Erkgrowth and Akt via [104 the]. Moreover,apoptotic De pathways Rosa et al. reviewed by the downregulation of the efﬁcacy of a combination therapy of docetaxel and zoledronate on hormone sensitive prostatethe anti cancer‐apoptotic cell line, protein LNCaP. ABcl combination‐2 [105]. of DTX and ZOL in hormone and drug refractory, PC-3 and DU-145 prostate cancer cells resulted in a signiﬁcant synergistic effect resulting4.2.2. Bisphosphonates from inhibited cell growth Conjugates via the apoptotic for the pathwaysTreatment by the of downregulationProstate Cancer of the anti-apoptotic protein Bcl-2 [105]. Boissier et al. prepared the BP‐conjugate of prostate cancer, as shown in Figure 11. 4.2.2.The Bisphosphonatesin vitro inhibitory Conjugates effects for the of Treatment four BPs of Prostate(ibandronate, Cancer zoledronate, risedronate, and clodronate)Boissier et al.and prepared three theBP‐ BP-conjugateconjugates of (NE prostate‐10244 cancer, (52), as NE shown‐58051 in Figure (53) 11 and. NE‐10790 (54)) The in vitro inhibitory effects of four BPs (ibandronate, zoledronate, risedronate, and clodronate)were evaluated. and three Zoledronate, BP-conjugates (NE-10244 ibandronate, (52), NE-58051 and compound (53) and NE-10790 52 were (54)) more effective than werecompound evaluated. 53 Zoledronate, and compound ibandronate, 54. Compound and compound 52 52inhibited were more tumor effective cell than invasion with an IC50 compoundvalue of 535 × and 10 compound−10 M. Compound 54. Compound 54 52inhibited inhibited tumortumor cell cell invasion invasion with an similar IC50 to that observed value of 5 × 10−10 M. Compound 54 inhibited tumor cell invasion similar to that observed withwith compound compound 52, whereas 52, whereas compound compound 53 did not show 53 did any not inhibitory show effect. any inhibitory Compound effect. Compound 5252 inhibited inhibited the growththe growth of the PC-3 of the prostate PC‐3 cancer prostate cell line cancer in vitro cell [106 line]. in vitro [106].

R: 52 N PO3H2 N 53 HO R H2O3P PO3H2

OH COOH N 54 FigureFigure 11. 11.Structures Structures of BP-conjugates of BP‐conjugates with anti-prostate with anti cancer‐prostate activity 52–54cancer. activity 52–54. 4.3. Lung Cancer 4.3.Lung Lung cancer Cancer is known as one of the leading causes of death worldwide [107,108]. In the UnitedLung cancer States in is 2018, known it was as reported one of that the lung leading cancer causes is accountable of death for 234,030 worldwide [107,108]. In new cases and 154,050 deaths. Tobacco smoking is one of the most widely recognized riskthe factor United contributing States in to 2018, lung cancer it was [109 reported]. Lung cancer that can lung be classiﬁed cancer intois accountable two types, for 234,030 new (1)cases non-small and 154,050 cell lung cancerdeaths. (NSCLC), Tobacco which smoking tends to is respond one of poorly the most to chemotherapy widely recognized risk fac‐ andtor radiationcontributing therapy; to and lung (2) cancer small cell [109]. lung cancer Lung (SCLC)—this cancer can group be classified is not common into two types, (1) non‐ and is categorized by high proliferation rate and also sensitivity to combined chemotherapy andsmall radiation cell lung therapy cancer in limited-stage (NSCLC), carcinoma which cases tends [109 ].to Bone respond metastases poorly development to chemotherapy and ra‐ isdiation 30–40% intherapy; patients withand NSCLC. (2) small The cell high mortalitylung cancer rate is (SCLC)—this predominantly a group result of is the not common and is difﬁcultiescategorized in the by early high diagnosis proliferation of bone metastases rate and and also the sensitivity high-metastatic to combined capability of chemotherapy and lungradiation cancer [ 107therapy]. A study in limited that was conducted‐stage carcinoma in Japan demonstrated cases [109]. that Bone bone metastasismetastases development is was present at the time of diagnosis in 48% of patients with stage four NSCLC and 40% of those30–40% with extensive-stagein patients with SCLC NSCLC. [110]. The The development high mortality of bone metastases rate is inpredominantly lung cancer a result of the candifficulties lead to SREs, in for the example, early pathologicdiagnosis fractures, of bone spinal metastases cord compression, and the hypercalcemia, high‐metastatic capability of andlung radiation cancer therapy [107]. A [111 study]. The that therapeutic was conducted approaches in to Japan treat bone demonstrated metastases are that bone metastasis still limited [112]. Hence, it is vital to develop treatment of bone metastases to inhibit SREswas [ 113present,114]. at the time of diagnosis in 48% of patients with stage four NSCLC and 40% of thoseIn the preclinicalwith extensive studies,‐ BPsstage anticancer SCLC effect [110]. against The NSCLC, development such as invasion, of bone an- metastases in lung giogenesis,cancer can inhibition lead to of tumorSREs, cell for proliferation, example, andpathologic micro-metastasis fractures, have been spinal reported. cord compression, hy‐ Moreover,percalcemia, preclinical and studiesradiation also therapy revealed that[111]. BPs The improved therapeutic the inhibitory approaches effects of to treat bone metas‐ EGFR-TKIs on NSCLC with EGFR mutation both in vitro and in vivo [112]. However, BPs tases are still limited [112]. Hence, it is vital to develop treatment of bone metastases to inhibit SREs [113,114]. In the preclinical studies, BPs anticancer effect against NSCLC, such as invasion, an‐ giogenesis, inhibition of tumor cell proliferation, and micro‐metastasis have been re‐ ported. Moreover, preclinical studies also revealed that BPs improved the inhibitory ef‐ fects of EGFR‐TKIs on NSCLC with EGFR mutation both in vitro and in vivo [112]. How‐ ever, BPs associated with nephrotoxicity, need observing and might require initial dose change and withholding of doses [108].

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Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 23 associated with nephrotoxicity, need observing and might require initial dose change and withholding of doses [108].

4.3.1. In Vitro StudiesStudies ofof BPsBPs inin LungLung CancerCancer BPs are powerful inhibitors ofof osteoclastic bonebone resorption andand havehave beenbeen commonly utilized inin thethe managementmanagement ofof hypercalcemia.hypercalcemia. Yano etet al.al. reported aa modelmodel ofof variousvarious organ metastasesmetastases withwith SCLCSCLC cellcell line,line, whichwhich includesincludes SBC-5,SBC‐5, inin NK3NK3 cell-depletedcell‐depleted SCIDSCID mice [114]. [114]. In In the the SBC SBC-5‐5 model, model, the the cell cell metastasizes metastasizes in inmultiple multiple organs, organs, which which include include the theliver, liver, lung, lung, kidney, kidney, and and bone, bone, resembling resembling characteristics characteristics of SCLC of SCLC in humans. in humans. A BP, A mi BP,‐ minodronatenodronate (YM529), (YM529), prevents prevents osteolytic osteolytic bone bone metastasis metastasis through through the prevention the prevention of bone of boneresorption resorption [114]. [Moreover,114]. Moreover, YM529 YM529 anticancer anticancer effects effectson different on different kinds of kinds cancer of cells cancer in cellsvitro inand vitro in vivoand havein vivo beenhave reported been reported[112]. However, [112]. YM529 However, did YM529 not extend did the not survival extend theof cancer survival‐bearing of cancer-bearing mice because mice of visceral because metastasis of visceral [114]. metastasis Furthermore, [114]. Furthermore,research that researchdemonstrated that demonstrated the impacts of the YM529 impacts on ofNSCLC YM529 has on been NSCLC limited. has been Therefore, limited. the Therefore, study of theYM529 study displayed of YM529 its anticancer displayed activity its anticancer on NSCLC activity cell onlines NSCLC in vitro cell and lines promptedin vitro apopand‐ promptedtosis and the apoptosis G1 arrest and cell the cycle G1 arrest via down cell cycle‐regulation via down-regulation of phosphorylation of phosphorylation of ERK1/2 [115]. of ERK1/2 [115]. 4.3.2. Bisphosphonates Conjugates for the Treatment of Lung Cancer 4.3.2. Bisphosphonates Conjugates for the Treatment of Lung Cancer An ideal chemotherapeutic drug designed for the treatment of bone metastases An ideal chemotherapeutic drug designed for the treatment of bone metastases should should specifically target the cancer cells in the bone, resulting in cytotoxicity to malignant specifically target the cancer cells in the bone, resulting in cytotoxicity to malignant cells cells while sparing normal cells, particularly in the bone marrow. Various efforts have while sparing normal cells, particularly in the bone marrow. Various efforts have been madebeen made to utilize to utilize BPs for BPs the for transportation/delivery the transportation/delivery of radiotherapy of radiotherapy and/or and/or chemotherapy chemo‐ basedtherapy on based their on known their affinityknown foraffinity bone for [116 bone]. El-Mabhouh [116]. El‐Mabhouh et al. developed et al. developed a drug a deliv- drug erydelivery system system using using an improved an improved BP (Figure BP (Figure 12) as 12) a bone-seekingas a bone‐seeking carrier carrier to transport to transport the chemotherapeuticthe chemotherapeutic drug, drug, gemcitabine gemcitabine (Gemzar) (Gemzar) (55) (55) to bone to bone lesions lesions while while preventing preventing the normalthe normal tissues tissues from from the toxicthe toxic effect effect of the of the drugs. drugs. Compound Compound 55 is55 commonly is commonly utilized utilized as anas an adjuvant adjuvant or or palliative palliative drug drug for for NSCLC, NSCLC, bladder, bladder, breast breast cancer, cancer, and and pancreatic. pancreatic. More-More‐ over, compoundcompound 5555 exhibitsexhibits aa widewide efficacyefficacy profileprofile withwith otherother formsforms ofof cancer.cancer. ItsIts broadbroad therapeutictherapeutic profileprofile makesmakes itit anan attractiveattractive antineoplasticantineoplastic agentagent forfor targetedtargeted chemotherapychemotherapy applications [117]. Schott et al. prepared a new antimetabolite‐BPs conjugate, 57. The IC50 applications [117]. Schott et al. prepared a new antimetabolite-BPs conjugate, 57. The IC50 concentrationconcentration of thethe compoundcompound onon LewisLewis lunglung carcinomacarcinoma (LLC)(LLC) exhibitedexhibited anan incubationincubation time-dependenttime‐dependent growth inhibitioninhibition withwith higherhigher sensitivitysensitivity towardstowards thethe tumortumor cells.cells. TheThe cytotoxiccytotoxic activity of compoundcompound 57 waswas confirmedconfirmed byby thethe cellcell viabilityviability test.test. To reachreach thethe ICIC50 forfor LLC cells, incubation of 72 h was required since thethe incubationincubation of 2424 hh waswas notnot sufficientsufficient forfor thethe compound.compound. However, thethe fundamentalfundamental mechanismsmechanisms ofof thesethese promisingpromising novel antimetabolite-BPsantimetabolite‐BPs conjugates remain to be evaluated in future experiments [[118].118]. TableTable6 6 depict depict a a summarysummary ofof thethe mode of action action of of bisphosphonate bisphosphonate on on different different types types of ofcancer. cancer.

OH PO3HNa

NaHO P NH2 3

NH2 N NH O N X H2O3P O N N N O H O X O N O H2O3P HO O N 55 X HO O 56 H HO X 57 HO X X-F HO H Figure 12.12. Structures ofof promisingpromising BP-conjugatesBP‐conjugates withwith anti-lunganti‐lung cancercancer activityactivity 55–57.55–57.

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Table 6. Mode of action of BPs in breast, prostate, and lung cancer.

Types of Cancer Mode of Action of BPs References - Inhibits proliferation of breast cancer cells, inhibits FPPS of the mevalonate [119,120] pathway and inhibits GGPPS. Shows high afﬁnity to bone matrix hydroxyapatite breast cancer. [121] - Induces apoptosis by preventing ATP-dependent enzymes and prevents their [122] absorption capacity. - Prevents breast cancer cell adhesion to the bone in vitro. Breast - Inhibits the development and capability of cultured human breast cancer cells. [123] - Induces loss of cell capability and DNA fragmentation in MCF-7 cells. - Prevents recurrence in postmenopausal women only. [124] - In vitro, prevents tumor cell invasion, adhesion, migration, proliferation, and [125] induces tumor cell apoptosis. - Improves the capability of antineoplastic agents to prevent breast cancer cell [126] invasion. - Induces MCF-7 cell death and inhibit MCF-7 cell growth. [127] - Has exhibited to apply a direct cytostatic and pro-apoptotic impact on PCa cell lines in vitro. - Inhibits cell invasion and adhesion through a decrease of matrix [128] metalloproteinase appearance. - Prevents testosterone-prompted angiogenesis in a castrated animal model. Prostate - Prevents proliferation and induce apoptosis of prostate cancer cell lines in vitro. - In vitro studies of PC-3, LNCaP, and Du145 cell line, it prevents proliferation, [129] induces apoptosis, decreases cell viability, and causes cell-cycle arrest. - Can down-regulate the expression of Bcl-2. - Induce apoptosis in prostate cancers. [130] - Prevents proliferation markers, destroying the proliferation of tumors. - Prevents cell proliferation in SCLC and NSCLC cell lines. - Uses its anti-proliferative impact against NSCLC by the initiation of cellular [115,131] apoptosis via the small GTP-binding proteins related signal transduction pathway. - Prevents cancer cell cycle progression of NSCL carcinomas. [125] Lung - Induces cancer cell apoptosis in osteosarcoma, melanoma, and mesothelioma. - Improves cancer cell apoptosis, yields synergistic anticancer effects. [132] - Prevents the action of osteoclasts and induces osteoclast apoptosis. - Exhibits prevention of the mevalonate pathway, regulation of immune response, [133] and affects tumor signaling pathways and anti-angiogenesis.

5. Conclusion and Future Perspective Bisphosphonates have been utilized as therapeutic agents for the treatment of osteoporosis, Paget’s disease, bone pain related to metastatic disease, hypercalcemia of malignancy, in diagnostic nuclear medicine, and targeted radiotherapy [134]. Therefore, the need for anti-osteoporotic agents that can be used for a prolonged duration with significant safety and efﬁcacy is very important. To inhibit fractures in people with osteoporosis, BPs therapies that are effective in decreasing the risk of fractures and preventing osteoclast activity, inhibit osteoclast genesis and promote osteoclast apoptosis, are crucial. The results of vitamin D-bisphosphonate derivatives displayed stimulated bone matrix formation in dosages that did not increase calcium levels, indicating the existence of a therapeutic window for the treatment of bone diseases. The synthesized vitamin D-bisphosphonate compound prompted osteoid formation in vivo without elevating calcium levels. The bisphosphonate-conjugated estrogen displayed a preference profile in the bone due to its characteristic distribution pattern related to the natural estrogen. Therefore, bisphosphonate- Int. J. Mol. Sci. 2021, 22, 6869 16 of 21

conjugated estrogens have the potential to enhance patient compliance in estrogen treatment by decreasing the side effects and decreasing the frequency of drug administration. BPs have been successfully used for the treatment of cancer-related bone disease and in reducing pain and skeletal complications. The skeleton is the most prevalent site to be affected by metastatic cancer. Prostate, lung, and breast cancers are responsible for the majority of skeletal metastases. This reflects both the high incidence and relatively long clinical course of these tumors [37,41]. BPs have also been utilized to decrease pain in multiple myeloma and reduce skeletal complications and the metastatic bone phase of a variety of solid tumors, for example, lung, prostate, and breast cancer. Some studies revealed the added beneficial clinical effect of BPs in cancer patients is associated with their anticancer activity. Some of the derivatives showed strong cytotoxicity on multiple myeloma cell lines and decreased the number of viable cells in a dosage-dependent manner. DEBP-Pt with high hydroxyapatite affinity showed bone resorption inhibitory effect and anticancer activity. The results suggest the potential of DEBP-Pt as a drug for metastatic bone cancer. Some BP-conjugates such as compound 47 could be delivered to the bone and demonstrated anticancer activity and prolonged survival, making it a promising therapy for TIBD. Compound 47 is one of the BP-conjugates in clinical trials. 5-Fluoro- 20-deoxyuridine-alendronate treatment in mice with bone metastases resulted in bone formation, inhibition of bone resorption, and inhibition of tumor growth. Long-term in vivo studies are warranted to determine the safety, effects on bone modeling and remodeling, and to further elucidate the cellular and molecular mechanisms leading to inhibition of tumor growth [88]. Linking BPs with conventional therapies for improved targeting and cytotoxicity in the case of cancer cells has the potential to provide synergistic effects for improving many treatments.

Author Contributions: Authors contributed equally to work reported. The authors approved the final version of the manuscript. Funding: This research was funded by the National Research Foundation South Africa, Sasol Inzalo Foundation, South African Medical Research Foundation, and Govan Mbeki Research and Development Centre (GMRDC), University of Fort Hare. Institutional Review Board Statement: Not applicable. The study does not involve the use of humans or animals. Informed Consent Statement: Not applicable. The study does not involve the use of humans. Conflicts of Interest: The authors declare no conflict of interest.

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