*Article* **Clinical Utility of Extracorporeal Shock Wave Therapy on Hypertrophic Scars of the Hand Caused by Burn Injury: A Prospective, Randomized, Double-Blinded Study**

#### **So Young Joo 1,**†**, Seung Yeol Lee 2,**†**, Yoon Soo Cho 1 and Cheong Hoon Seo 1,\***

Received: 8 April 2020; Accepted: 5 May 2020; Published: 7 May 2020

**Abstract:** Postburn hypertrophic scarring is a common complication in burn injuries to the hands, often associated with impaired hand function. We evaluated the effects of extracorporeal shock wave therapy (ESWT), compared to a sham stimulation therapy, on hypertrophic scars of the hand caused by burn injury and investigated its effects on hand function. This was a double-blinded, randomized, controlled trial of 48 patients with a burn to their dominant right hand. The parameters of ESWT were as follows: energy flux density, 0.05–0.30 mJ/mm2; frequency, 4 Hz; 1000 to 2000 impulses per treatment; four treatments, once a week for four weeks. The outcomes measured were as follows: a 10-point visual analogue scale pain score; Vancouver scar scale for scar vascularity, height, pliability and pigmentation; ultrasound measurement of scar thickness; Jebsen−Taylor hand function test; grip strength; Perdue pegboard test; and the Michigan hand outcomes questionnaire. The change in the score from baseline to post-treatment was compared between the two groups.  The other measured outcomes were not different between the two groups. ESWT is effective in decreasing pain, suppressing hypertrophic scarring, and improving hand function.

**Keywords:** extracorporeal shock wave therapy; hypertrophic scar; burn; hand function

#### **1. Introduction**

Hypertrophic scarring is a common complication after a burn. Hypertrophic scars result from excessive tissue formation during the wound healing process [1]. The mechanisms underlying hypertrophic scar formation include exaggerated inflammation, prolonged re-epithelialization, excessive extracellular matrix production, and reduced apoptosis [2]. Both types of hypertrophic scars, those which are raised and inflexible [3] and those which are characterized by traction on surrounding tissues, can result in functional limitations [4]. Common complications after burn injuries to the hands include decreased hand performance, sensory impairment, and scar contracture [5]. Hand complications such as these may impair performance of the activities of daily life. Nonsurgical treatments of postburn hypertrophic scars include intralesional corticosteroid injection, laser therapy, compression therapy, and silicone therapy. The outcomes of these nonsurgical treatments of hypertrophic scars on the hands, however, have not yielded satisfactory outcomes. Recently, there has been increased focus on shortening the healing time and improving hand function to reduce the risk of hypertrophic scarring.

Extracorporeal shock wave therapy (ESWT) is effective in the treatment of orthopedic diseases and neuropathic pain and has also been used in regenerative medicine of the skin [6], with neoangiogenesis and anti-inflammatory effects having been reported [7]. The regeneration effects of ESWT have been demonstrated in improving the healing of burn wounds [8,9] as well as in decreasing hypertrophic scarring after a burn [10]. ESWT may also improve the pliability and the subjectively evaluated appearance of postburn scar contracture [11]. Studies in our own institution found ESWT to be useful for burn-associated pain and pruritus and demonstrated that ESWT can suppress the hypertrophic scarring. The anti-fibrotic effects of ESWT can likely be attributed to its molecular effect on hypertrophic scars [1,12,13]. However, the effects of ESWT on hypertrophic scarring of the hand and on hand function have not been evaluated to date. Therefore, this research aimed to evaluate the effectiveness of ESWT on hypertrophic scars and on the hand functions affected by such scarring.

#### **2. Experimental Section**

#### *2.1. Methods*

#### Study Design and Statement of Ethics

This was a double-blinded, randomized, controlled trial of 48 patients (45 male, 3 female) recruited from the Department of Rehabilitation Medicine, Hangang Sacred Heart Hospital, Korea, between November 2019 and April 2020. Our study was approved by the Ethics Committee of the Hangang Sacred Heart Hospital (HG2018-047) and registered in ClinicalTrials (NCT04138355). All patients provided written informed consent.

#### *2.2. Study Group*

Each of the 48 patients enrolled in the study were ≥18 years old and had sustained a deep partial-thickness (second-degree) burn or a full thickness (third-degree) burn involving only the right hand, which had been treated with a split-thickness skin graft (STSG) after the thermal injury, <6 months prior to enrollment. At the time of the study, all the patients were in the re-epithelialization phase of wound healing and had been transferred to the rehabilitation department, after acute burn treatment, to improve hand and wrist stiffness due to painful retracting scarring. The criteria for exclusion from the study were as follows: left-hand dominance; musculoskeletal diseases (fracture, amputation, rheumatoid arthritis, and degenerative joint diseases) of the right dominant hand; pregnancy; or potential for additional damage to the skin if exposed to ESWT and conventional occupational therapy.

The first 50 burn patients who met our inclusion/exclusion criteria were randomly allocated, using a computer program, to either the ESWT (n = 25) group or to the sham (n = 25) group. While all participants in the sham group completed the trial protocol, two patients in the ESWT group dropped out of the study because they did not want to undergo serial evaluations. Thus, 23 patients were ultimately included in the ESWT group, and 25 were included in the sham group (Figure 1).

**Figure 1.** Diagram for subject enrollment, allocation, and follow-up.

#### *2.3. Intervention*

Patients in both groups received standard rehabilitation treatment for burn injuries to the hands, including medication, scar lubrication, massage therapy to the scars, and occupational therapy to improve hand function. Occupational therapy treatment consisted of 20 sessions (30 min per day, five days a week) for four weeks. Every effort was made to provide the same exercise rehabilitation for both groups and to adapt the level of difficulty of the exercises prescribed to each patient's performance. Patients received one ESWT or sham treatment session per week for four weeks.

Patients in the ESWT group were asked to select the most hypertrophic and retracting scar on their dominant right hand for treatment. ESWT was conducted using the Duolith SD-1 device (StorzMedical, Tägerwilen, Switzerland), with an electromagnetic cylindrical coil source used to focus the shock wave (Figure 2). ESWT was performed around the primary treatment site, at an intensity of 100 impulses/cm2, an energy flux density (EFD) of 0.05 to 0.30 mJ/mm2, and a frequency of 4 Hz. Between 1000 to 2000 impulses were administered per session, for four sessions held at one week intervals. In the sham group, the same shock wave equipment, with the same-shaped adapter, as in the experimental group was used, but with no energy emitted (Figure 3).

**Figure 2.** The extracorporeal shock wave therapy was administered to burn patients. The administered shock wave dose was 100 impulses/cm2 at 0.05 to 0.30 mJ/mm2 with a total of 1000 to 2000 impulses per treatment.

**Figure 3.** Sham adapter: (**A**) inside view; (**B**) outside view.

#### *2.4. Outcome Measures*

In order to evaluate the effect of ESWT, we compared the change in the severity of pain, scar thickness, and hand function between the ESWT and sham groups, from baseline measures taken immediately before the intervention and measures taken immediately after session 4. The 10-point visual analogue scale (VAS) was used to measure self-reported pain severity, with ratings from 0 (no pain) to 10 (unbearable pain). The Vancouver scar scale (VSS), an observer-dependent scale of the macroscopic appearance of scarring, was used to measure changes in the scar over the four week period of treatment [14]. The VSS consists of four parameters (vascularity, height, pliability, and pigmentation), scored in total on a 14-point scale, with a higher score indicative of a better outcome. The thickness of the

scar was objectively quantified by ultrasound (128 BW1 US system, Medison, Korea). Grip and pinch strength were quantified using a hand-held dynamometer (Lafayette Instrument, USA). The Michigan hand outcomes questionnaire (MHQ) was used to assess a patient's perception of hand function on a scale of 0–100, with a higher score indicative of a better outcome [15]. The Jebsen−Taylor hand function test (JTT) was used to measure the performance speed of standardized tasks. The JTT consists of seven subtests, each scored on a scale of 0–15, with a higher score indicative of better hand function [16]. In the Perdue pegboard test (PPT), motor function is measured by the number of pins that can be placed in the board (along two parallel rows with 25 holes each) in 30 s, and dexterity is measured by the number of pins, washers, and collars that can be assembled in 60 s. Scores were evaluated for the affected right (dominant) hand and both hands [17]. Outcome measurements and data analyses were performed by a trained and blinded outcome assessor who was not involved in the intervention. Possible complications (pain, ecchymosis, skin abrasion, and swelling) were observed.

#### *2.5. Statistical Analysis*

Statistical analyses were performed using SPSS, version 23 (IBM Corp., Armonk, NY, USA). Fisher's exact test was used to evaluate the homogeneity of the distribution of sex and burn types between the two groups before treatment, and the independent t-test was used to evaluate the homogeneity of distribution of the total burn surface area (TBSA), scar thickness, total VSS, dominant-hand and bilateral PPT scores, and the score for picking up small objects subtest of the JTT. A between-group *p*-value < 0.05 was deemed significant. The pre- to post-treatment scores were evaluated between the two groups using the Mann−Whitney or independent t-test, as appropriate for the variable type and distribution, with a *p*-value < 0.05 deemed significant.

#### **3. Results**

There were no differences between the two groups in terms of demographic and clinical characteristic before treatment (*p* > 0.05 for all comparisons; Table 1). There were no differences in the measured outcomes before treatment between the two groups (*p* > 0.05 for all comparisons; Table 2).

**Table 1.** Baseline characteristics of the study group.

 Values are presented as mean ± standard deviation. The *p*-values were calculated using Fisher's exact test, the Mann−Whitney test, or Student's t-test as appropriate.

**Table 2.** Outcome measures at baseline.

VAS, visual analogue scale; ESWT, extracorporeal shock wave therapy. Values are presented as the mean ± standard deviation. The *p*-values were calculated using Fisher's exact test, the Mann−Whitney test, or Student's t-test as appropriate.

More reductions were found in the ESWT group than in the sham group for the pre- to post-treatment change in the VAS score (*p* = 0.001) and vascularity VSS score (*p* = 0.0015) (Table 3). There was a slight decrease in the pre- to post-treatment change of scar thickness (*p* = 0.018) for the ESWT group over the sham group (Table 3). However, there were no significant differences in the change score between the two groups for scar pigmentation (*p* = 0.19), pliability (*p* = 0.78), height (*p* = 0.66), and the total VSS score (*p* = 0.19; Table 3). Similarly, grip strength improved in both groups, without a difference in the change scores between the two groups (grasp, *p* = 0.99; lateral pinch, *p* = 0.46; and tip pinch, *p* = 0.26; Table 3). For the JTT, the change scores were significantly greater for the ESWT than the sham group for the subtasks of simulated card-turning (*p* = 0.02) and picking up small objects (*p* = 0.004), with no between-group differences in the change score for the remaining subtasks (writing, *p* = 0.12; stacking checkers, *p* = 0.15; simulated feeding, *p* = 0.99; picking up large light objects, *p* = 0.16; and picking up large heavy objects, *p* = 0.90; Table 3). For the PPT, there were no significant differences in the change scores for the affected hand (*p* = 0.12), both hands (*p* = 0.97), and assembly (*p* = 0.44; Table 3). Furthermore, there were no between-group differences in any of the subscores of the MHQ (Table 3): function (*p* = 0.17); activities of daily living, ADL (*p* = 0.51); work (*p* = 0.12); pain (*p* = 0.45); esthetics (*p* = 0.06); and satisfaction (*p* = 0.15).

**Table 3.** Change score (pre- to post-treatment) on measured outcomes.

VAS, visual analogue scale; ESWT, extracorporeal shock wave therapy. Values are presented as the mean ± standard deviation. The *p*-values for between-group differences were calculated using the Mann−Whitney test (\*\*, *p* < 0.05) and independent t-test (\*, *p* < 0.05), as appropriate.

#### **4. Discussion**

We evaluated the effectiveness of ESWT on the hypertrophic scars of hand burns that required STSG. Therapeutic effects were evaluated by the change in scar pain, thickness, esthetic characteristics, and hand function after a four week period of treatment. The ESWT treatment of hypertrophic scarring after a burn injury to the hands provided significant benefits in improving hand function, decreasing pain, and suppressing hypertrophic scar growth.

Cho et al. [12] have reported on the effects of ESWT applied to the area of most severe pain after complete epithelialization on pain outcomes (ESWT dose: EFD, 0.05 to 0.15 mJ/mm2; frequency, 4 Hz; and three sessions at one week intervals). The authors proposed that measured improvement in pain control with ESWT had resulted from a decrease in the number of unmyelinated nerve fibers; inhibition of nociceptors from repeated stimulation; regulation of neuroinflammatory molecules, such as substance P; and increased blood flow, facilitating tissue regeneration via an increase in endothelial nitric oxide synthase (eNOS). Joo et el. applied ESWT to burn scars with severe pruritus, using the same protocol as described by Cho et al., and found ESWT to be effective in reducing the severity of pruritus and improving ADLs [13]. Epithelial–mesenchymal transition (EMT), in which epithelial cells lose the inclination to reproduce, is known to have a role in fibrogenesis during hypertrophic scarring [18]. Seo et el. [1] identified that ESWT (EFD of 0.03, 0.1, and 0.3 mJ/mm2; a frequency of 4 Hz; and a volume of 1000 pulses) suppressed EMT by inhibiting the EMT inducer [1]. In our study, we used an intensity and frequency of ESWT previously shown to have a therapeutic effect for burns. Previous research has shown the effectiveness of low dose ESWT (0.1 mJ/mm2) in suppressing hypertrophic scar formation through inhibition of α-SMA expression [19,20]. Meirer et al. reported that ESWT, applied to partial-thickness burns, three and seven days after the burn injury (1500 impulses at 0.11 mJ/mm2), reduced the need for surgery and decreased scar formation during the six month follow-up period [21].

In our study, we also identified a significant benefit of ESWT in improving vascularization of the scar tissue, measured using the VSS. Previous research has investigated the mechanisms underlying the mechanosensitive feedback between ESWT and stimulated cells [22]. Nitric oxide (NO) production contributes to an increase in perfusion, with the improved blood supply preventing ischemia [22,23]. The accelerated epithelialization after ESWT can be explained by multiple factors, including neovascularization, increased granulation, and increased epithelial cell proliferation in the burn wound [8,9,24]. High-energy ESWT (0.3 mJ/mm2, frequency 4 Hz) increased the perfusion at remote sites via an increase of the NO and of vascular endothelial growth factor (VEGF) [25]. Davis et al. also demonstrated that the application of ESWT at 1 h postinjury significantly blunted the activity of inflammatory cells [26], although in clinical practice, hyperemia may occur at the site of ESWT due to an increase in local blood flow [6,13]. Overall, decreasing the healing time would be an important factor in reducing the risk of undesirable scarring and decreasing pain.

ESWT is known to be effective for reducing pain and improving function on orthopedic injuries [27]. Substance P may play a role in pain mediation in small unmyelinated C fibers. An intradermal injection of substance P causes a flare-up in pain, swelling, and pruritus. On the other hand, calcitonin gene-related peptide (CGRP) is a well-known marker of unmyelinated C fibers and thinly myelinated Aδ fibers, both of which are important in nociception. The loss of nerve fibers and the depletion of neuropeptides with ESWT might be effective in decreasing pain. A low dose of ESWT (0.08 mJ/mm2, frequency 4 Hz) applied to a knee as a treatment for osteoarthritis reduced neuropeptide expression, which was associated with improved pain and gait function [28]. More substance P and CGRP are observed in painful hypertrophic scars compared to normal skin [29]. This evidence reinforces the idea that the pain-control effect of ESWT is likely to be mediated by its effect on pain mediators, and substance P and CGRP more specifically. The clinical benefits of ESWT have been reported for a range of orthopedic disorders and associated with self-reported improvements in function and quality of life [30–32].

ESWT has been widely used in treatment of various tendinopathies in upper limbs. Steroids limit tenocyte function by reducing proteoglycan and collagen synthesis, which reduce mechanical strength. Unlike steroid therapy, ESWT studies have shown that ESWT affects tenocyte regeneration, leading to an improvement in muscle strength. The effectiveness of ESWT compared with that of local steroid injections showed that ESWT had favorable effects on resolution of pain and improvement of grip strength [33,34]. In our study, we identified improvements in grip strength with ESWT, as well as improvement in simulated card-turning and the picking up of small objects. This improved function might be related to improvements in the pliability of the scar because of ESWT breaking down collagen fibers to induce scar remodeling. The increased pliability of the scars with ESWT, with a less evident effect on coloration, has already been reported [11]. A previous study [4] has also indicated that unfocused application of shock waves (EFD, 0.13 mJ/mm2; frequency, 6 Hz; two sessions per week for five weeks) was effective in significantly improving VSS scores, scarring pain, and the range of movement of the hand. These clinical benefits of ESWT were thought to be mediated by stimulation of dermal fibroblasts, neoangiogenesis, and improvement in the parallel organization of collagen fibers within the skin. Similarly, a beneficial effect of ESWT has been reported for the treatment of trigger finger complaints, resulting from enhanced repair and healing of the degenerated tendon [34]. Overall, as pain, pruritus, and decreased function negatively impact the quality of life after a burn, ESWT may have a global effect in improving quality of life after burn injuries [35].

This study requires cautious interpretation of the data, for reasons of small sample size, a short follow-up period, and the absence of detailed measurement of range of motion in the affected hand. Future studies with a longer time frame and more detailed assessment are needed to confirm our findings. Continued basic research into the mechanisms underlying the clinical effects of ESWT are needed to determine optimal parameters for the clinical management of hypertrophic scars.

#### **5. Conclusions**

In making the comparison of ESWT with a sham stimulation therapy for the treatment of hypertrophic scarring of the hand after a burn injury, we identified a clinically beneficial effect of ESWT in promoting hand function, improving scarring, and alleviating scar-related pain. This indicates the clinical usefulness of the intensity and frequency parameters of ESWT that we used in our study. It is important to point out that the optimal frequency and intensity of ESWT for the treatment of hypertrophic scars of the hands after a burn remain to be determined. Our findings, however, do provide experimenters with the option to use ESWT for its potential to improve the management and treatment of hand burn scars after STSG.

**Author Contributions:** C.H.S. contributed as corresponding author of this work. S.Y.J. and S.Y.L. contributed equally to the conception, design, data analysis, and interpretation of results. Y.S.C. contributed to revising the article critically for important intellectual content. All authors contributed to the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2018R1C1B6008584) and the Soonchunhyang University Research Fund.

**Conflicts of Interest:** The authors declare no potential conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**3. Results and Discussion**

As we consider the WBLA, the detailed electronic structure of the leads is not important for the description of the transport properties of the GNR. We then fix the coupling strength between the GNR and the leads by the frequency-independent resonance width *Γα* = *tC*/10 corresponding to a weak-coupling regime where the WBLA is a good approximation [44–47]. This is further justified in typical transport setups where the bandwidth of the leads is sufficiently large (e.g., gold electrodes) compared to the applied bias voltage. As we wish to preserve the charge neutrality of the GNR in equilibrium, we set the chemical potential to *μ* = 0. The global equilibrium temperature is set by (*k*B*T*)−1 = 100*t* −1 *C*(*T* = 313 K).

### *3.1. Response to a dc Drive*

It is instructive to first study the current correlations in GNRs without disorder. Figure 2 shows the current cross-correlations of undisordered AGNRs and ZGNRs of various lengths and *time-independent* bias voltages. We can make many general observations from the data:

**Figure 2.** Absolute value of the current cross-correlation *C*(×) (*t* + *τ*, *t*) at long observation times *t* → ∞ for various undisordered GNR samples with varying bias voltage and the consequent Fourier spectra: (**a**) AGNR of various lengths; (**b**) ZGNR of various lengths; (**c**) the low-frequency region of the Fourier transform of (**a**) for fixed bias voltage *VL* = −*VR* = *tC*/2 (the inset shows the full frequency range); and (**d**) the low-frequency region of the Fourier transform of (**b**) for fixed bias voltage *VL* = −*VR* = *tC*/2 (the inset shows the full frequency range).


*4.3. Asymptotic E*ffi*ciency*

The asymptotic efficiency of *M*-estimates is numerically computed as follows:

$$\varepsilon f f(T\_n) = \frac{Var(T\_{ML})}{Var(T\_n)} = \frac{\left(\int \psi'(\mathbf{x}) p(\mathbf{x}) \,d\mathbf{x}\right)^2}{I(p) \int \psi^2(\mathbf{x}) p(\mathbf{x}) \,d\mathbf{x}}.$$

.


**2.1 Histoplasmosis treatment in immunocompetent host**

In immunocompetent individuals, the disease is usually mild, asymptomatic, or experiences a self-limiting condition with nonspecific symptoms (fever, malaise, chills, weight loss, night sweats, respiratory, rheumatologic, or gastrointestinal symptoms) and may require only symptomatic measures [7, 9, 11]. For most individuals, signs typically resolve without intervention, and only 5% of *Histoplasma* infections are estimated to require clinical management [11, 14].

Disseminated disease although more frequent among immunocompromised patients can be seen occasionally even in immunocompetent patients, when they have traveled in endemic county and have been infected with a large quantity of *H capsulatum*, or have been exposed to a highly virulent strain.

Antifungal treatment is indicated for certain forms of histoplasmosis, including acute moderate to severe histoplasmosis, chronic disease, and extrapulmonary histoplasmosis. The Infectious Diseases Society of America (IDSA) guidelines state that antifungals could be considered for patients with symptoms lasting >1 month [7]. In **Table 1**, we summarized histoplasmosis treatment considering the immune status and disease severity in acute and chronic forms of histoplasmosis.

#### **Table 1.**

*Treatment in histoplasmosis considering the immune status and disease severity in acute and chronic histoplasmosis [7, 11, 12, 15].*

In vitro activities of current antifungal drugs (the polyene, azole, and echinocandin classes) used clinically have been established for dimorphic fungi [11]. Antifungal treatment is generally not required in mild to moderate diseases if they do not have symptoms for more than four weeks. The initial therapy should be itraconazole, given as a loading dose (200 mg orally three times daily for the first three days) followed by a maintenance dose (200 mg orally once or twice daily) for 6 to 12 weeks [10].

For moderate form to severe progressive disseminated histoplasmosis cases, the IDSA guidelines recommend initial amphotericin B treatment (liposomal amphotericin B dosed 3 mg/kg/day intravenously or amphotericin B lipid complex dosed 5 mg/kg/day intravenously or amphotericin B deoxycholate dosed 0.7 to 1 mg/kg/ day intravenously) followed by itraconazole (200 mg orally three times daily for the first three days, then 200 mg orally twice daily) for a minimum of 12 weeks up to an additional three months [2, 7, 11].

Antifungal treatment in non-immunosuppressed patients is suggested for at least six months, although the severity and site of the disease need to be considered before determining the duration of therapy [11].

In patients with severe dyspnea, hypoxemia, and/or development of acute respiratory distress syndrome, the addition of methylprednisolone (0.5 to 1.0 mg/kg/d intravenously) for one to two weeks has been used in some patients with clinical benefit [16, 17].

All patients with chronic pulmonary histoplasmosis should be treated due to most patients` progressive loss of pulmonary function. Oral itraconazole is given as a loading dose (200 mg orally three times daily for the first three days) followed by a maintenance dose (200 mg orally once or twice daily) for at least one year until 24 months because of the substantial risk of relapse (after treatment stopped up to 10 to 20% of patients with chronic pulmonary histoplasmosis could relapse within two years of stopping therapy) [7, 18]. Chronic cavitary pulmonary histoplasmosis developed after the acute form is marked by low-grade chronic symptoms, development of persistent cavitation, pulmonary fibrosis and pulmonary insufficiency [19].

In chronic pulmonary histoplasmosis, the cavities should be monitored with chest radiography, and if they persist over a 2- to 4-month period, antimicrobial treatment is recommended, especially if there are also symptoms [19].

Chest radiography should be performed every six months for the first year and then annually for four years to monitor for relapse. Without therapy, pulmonary function progressively declines to respiratory insufficiency, the disease will progress, and the patients will die [7, 11, 17–19].

More recent series have emphasized that pulmonary nodules are a more common manifestation of chronic histoplasma infection that can persist a long time and should be differentiated from symptomatic patients who have multiple diffuse nodules and require acute pulmonary histoplasmosis treatment [7, 11].

## **2.2 Histoplasmosis treatment in immunocompromised patients**

An immunocompromised state (due to HIV or other sources of immunosuppression) increases the risk of systemic infection with histoplasmosis. In addition, the immune system dysregulation in autoimmunity and immunosuppressive medications increases serious infection risk as histoplasmosis [17, 20].

After invading the lungs, the infection progressively spreads to other organs. Immunocompromised patients are 10 to 15 times more likely to develop a disseminated form of the disease [7, 20, 21]. The disseminated disease is often fatal if it is not promptly recognized, particularly in patients with HIV infection (histoplasmosis is an AIDSdefining condition), stem cell or solid organ transplantation receivers, and requires a high index of suspicion for timely diagnosis and treatment. In HIV-infected patients, symptomatic illness has been observed in 55% of cases of primary histoplasmosis, and progressive disseminated histoplasmosis occurred in 97–100% of cases of symptomatic histoplasmosis before the availability of highly active antiretroviral therapy [6].

Severely immunocompromised patients often present acutely with fever, pancytopenia, severe respiratory distress, circulatory shock, coagulopathy, and multiorgan failure involving the liver and kidneys. In these patients, histoplasmosis is a life-threatening condition that may require a multi-disciplinary approach to care [20]. Patients with mild immune deficiencies produced by several factors (older age, advanced age, alcoholism, diabetes *mellitus*, solid tumors, corticotherapy, or lymphomas) are more prone to develop chronic histoplasmosis [19].

Histoplasmosis in the immunocompromised host has some particularities. One of the more severe forms of the disease, disseminated histoplasmosis, is a progressive extrapulmonary infection often seen in immunocompromised patients (e.g., AIDS, those on immunosuppressive medications, etc.) or at extremes of age.


**2. Synthesis of tritium-labeled BRs**

#### **2.1. BRs with very high SA of tritium (~99 Ci/mmol)**

For binding assays, study aimed at ligand-receptor-activity relationship is a high specific activity (SA), a bottom line requirement. The SA of such radio-labeled drugs need to be in scale of tenths of Ci/mmol. This critical precondition used to be an obstacle in the way of BR's studies for decades. The state-of-art strategy for such a labeling was reported by Marek et al. [14]. The methodology yields tritium-labeled BRs bearing a very high SA of 99.4 and 98 Ci/mmol (approx. 3.4 tritium enrichment per molecule), respectively. Convenient, a sixstep synthetic sequence starting with the brassinosteroid *to be labeled* provides the desired tritium-multi-labeled product in sufficient yield (up to 40 mCi) with satisfactory radiochemical purity (>97%). The work is focused on the 24-[3 H]epibrassinolide [3 H]-**1** and 24-[3 H]epicastasterone [3 H]-**2**, both labeled in the side chain of BR skeleton on positions of carbon C-24, -25, -26, and -27 (**Figure 2**). The labeling strategy is designed to employ a radio-labeling step at the later stage of the synthetic sequence.

In 1998, Seto et al. described a fairly elegant strategy for the deuterium-multi-labeling of brassinolide in its side-chain [15]. The five-step reaction sequence was started by full protection of hydroxyl groups on the BR. The C-25 carbon was oxidized by freshly generated trifluoromethyldioxirane (TFD) that yielded appropriate hydroxy derivative. Its consecutive dehydration led to a mixture of Δ25(26) and Δ24(25) regiomers in the 65:35 ratio that was possible to separate after deprotection. The deuteration of Δ25(26) regioisomer by deuterium gas catalyzed by Pd/C (1 atm, 25°C, 1 h) yielded [24, 25, 26, 27-2 H]brassinolide with 60% deuterium enrichment calculated from MS data. The ratio of the individual multi-deuterated species in the cluster was 2 H2 : 2 H3 : 2 H4 : 2 H6 : 2 H7 = 3:8:14:15:60. The basic idea of this methodology for usage at labeling with radioactive isotope tritium was waiting almost for two decades—then 24-[3 H] epiBL (**1**) and 24-[3 H]epiCS (**2**) was synthesized [14].

The protocol of Seto et al. paved the way for the synthesis of an unsaturated precursor for the intended synthesis of 3 H-labeled 24-epiCS [15, 16]. First, the 2,3-22,23-bisisopropylidene derivative **4** was prepared in a 96% yield by a reaction of 24-epiCS (**2**) with 2,2-dimethoxypropane catalyzed by *p*-toluenesulfonic acid [14]. Having 2,3,22,23-diisopropylidene-24-epicastasterone **4** available, the TFD hydroxylation of C-25 carbon with this particular derivative was studied. Unfortunately, the desired hydroxylation was accompanied by oxidation of alcohol C-3 affording appropriate hydroxyketone. Hence, more resistant 2,3,22,23-tetra-O-acetyl-24-epiCS was prepared. However, its C-25 hydroxylation did not proceed at all under the conditions used for the hydroxylation of **4**. Finally, 2,3-di-O-acetyl-22,23-isopropylidene-24-epicastasterone (**6**) was prepared which upon TFD hydroxylation at low temperature gave 2,3-di-O-acetyl-22,23-isopropylidene-25-hydroxy-24-epiCS (**7**) in 58% yield (**Figure 2**). The dehydration of **7** using thionyl chloride in pyridine at 0°C for 30 min afforded a mixture of prevailing Recent Developments in a Radio-labeling of Brassinosteroids http://dx.doi.org/10.5772/intechopen.68584 15

This chapter is engaged in the recent developments in the synthesis of 3

14 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

For binding assays, study aimed at ligand-receptor-activity relationship is a high specific activity (SA), a bottom line requirement. The SA of such radio-labeled drugs need to be in scale of tenths of Ci/mmol. This critical precondition used to be an obstacle in the way of BR's studies for decades. The state-of-art strategy for such a labeling was reported by Marek et al. [14]. The methodology yields tritium-labeled BRs bearing a very high SA of 99.4 and 98 Ci/mmol (approx. 3.4 tritium enrichment per molecule), respectively. Convenient, a sixstep synthetic sequence starting with the brassinosteroid *to be labeled* provides the desired tritium-multi-labeled product in sufficient yield (up to 40 mCi) with satisfactory radiochemi-

In 1998, Seto et al. described a fairly elegant strategy for the deuterium-multi-labeling of brassinolide in its side-chain [15]. The five-step reaction sequence was started by full protection of hydroxyl groups on the BR. The C-25 carbon was oxidized by freshly generated trifluoromethyldioxirane (TFD) that yielded appropriate hydroxy derivative. Its consecutive dehydration led to a mixture of Δ25(26) and Δ24(25) regiomers in the 65:35 ratio that was possible to separate after deprotection. The deuteration of Δ25(26) regioisomer by deuterium gas cata-

enrichment calculated from MS data. The ratio of the individual multi-deuterated species in

The protocol of Seto et al. paved the way for the synthesis of an unsaturated precursor for the

ative **4** was prepared in a 96% yield by a reaction of 24-epiCS (**2**) with 2,2-dimethoxypropane catalyzed by *p*-toluenesulfonic acid [14]. Having 2,3,22,23-diisopropylidene-24-epicastasterone **4** available, the TFD hydroxylation of C-25 carbon with this particular derivative was studied. Unfortunately, the desired hydroxylation was accompanied by oxidation of alcohol C-3 affording appropriate hydroxyketone. Hence, more resistant 2,3,22,23-tetra-O-acetyl-24-epiCS was prepared. However, its C-25 hydroxylation did not proceed at all under the conditions used for the hydroxylation of **4**. Finally, 2,3-di-O-acetyl-22,23-isopropylidene-24-epicastasterone (**6**) was prepared which upon TFD hydroxylation at low temperature gave 2,3-di-O-acetyl-22,23-isopropylidene-25-hydroxy-24-epiCS (**7**) in 58% yield (**Figure 2**). The dehydration of **7** using thionyl chloride in pyridine at 0°C for 30 min afforded a mixture of prevailing

at labeling with radioactive isotope tritium was waiting almost for two decades—then 24-[3

H]epibrassinolide [3

H7 = 3:8:14:15:60. The basic idea of this methodology for usage

H-labeled 24-epiCS [15, 16]. First, the 2,3-22,23-bisisopropylidene deriv-

H]-**2**, both labeled in the side chain of BR skeleton on positions of carbon C-24,

labeled analogs of the brassinolide.

**2. Synthesis of tritium-labeled BRs**

**2.1. BRs with very high SA of tritium (~99 Ci/mmol)**

cal purity (>97%). The work is focused on the 24-[3

lyzed by Pd/C (1 atm, 25°C, 1 h) yielded [24, 25, 26, 27-2

H]epiCS (**2**) was synthesized [14].

at the later stage of the synthetic sequence.

castasterone [3

the cluster was 2

epiBL (**1**) and 24-[3

intended synthesis of 3

H2 : 2 H3 : 2 H4 : 2 H6 : 2 H- and 14C-radio-

H]-**1** and 24-[3

H]brassinolide with 60% deuterium

H]epi-

H]

**Figure 2.** The successful approach for the synthesis of 24-[3 H]epiCS and 24-[3 H]epiBL with a high SA.

(22*R*,23*R*,24*R*)-2α,3α-diacetoxy-22,23-isopropylidenedioxy-24-methyl-5α-cholestan-25-ene-6 one (**8**) accompanied by its 24-ene regioisomer **9**. The separation of unsaturated regioisomers **8** and **9** from each other turned out to be infeasible using various conditions on high-performance liquid chromatography (HPLC). Importantly, it was possible to separate unsaturated derivatives **8** and **9** from 24-epicastasterone derivative **6** by HPLC. This fact eventually enabled the isolation of 261 mCi of (22*R*,23*R*,24*R*)-2α,3α-diacetoxy-22,23-isopropylidenedioxy-24-[24, 25, 26, 27-3 H]epicastasterone (**10**) after the catalytic tritiation of the mixture of the unsaturated derivatives **8** and **9** over Pd/C (10%) in ethyl acetate under carrier-free tritium gas (998 mbar) for 2 h. In one-pot synthesis, derivative **10** was deisopropylinated and deacetylated provided by HPLC purification, 40 mCi of 24-[24, 25, 26, 27-3 H]epicastasterone ([3 H]-**2**) with RCP >97% and SAMS = 99.4 Ci/mmol [14].

To get [24, 25, 26, 27-3 H]epibrassinolide ([3 H]-**1**), the Baeyer-Villiger oxidation on fully protected 24-[24, 25, 26, 27-3 H]epicastasterone **11** was carried out by freshly prepared chloroform solution of trifluoroperoxyacetic acid [30% H2 O2 (20 ml), trifluoroacetic acid (100 ml), CHCl3 (1 mL)] cooled to 0°C by an ice bath [14] . The de-isopropylidation of the (22*R*,23*R*,24*R*)-2α,3αdiacetoxy-22,23-isopropylidenedioxy-24-[24, 25, 26, 27-3 H]epiBL (**11**) was carried out by wet FeCl3 , afterwards full deacetylation was accomplished by methanol solution of CH3 ONa. This conditions was made possible to obtain 3.5 mCi of pure 24-[24, 25, 26, 27-3 H]-epiBL ([3 H]-**1**) with RCP > 97% and SAMS = 98 Ci/mmol. The 3 H NMR spectra of both [3 H]-**1** and [3 H]-**2** show tritium signals in C-25, C-26, and -27, and is in accordance with the determined SA, indicating 3.4 tritium atoms per molecule.

#### *2.1.1. Stability of BRs possessing very high SA*

The free BRs with a high SA are extremely sensitive to radiolysis if stored improperly. Authors reported one representative example—when a sample was evaporated to dryness and used for the NMR analysis (DMSO-*d6* ), the signals in 3 H NMR spectrum were difficult to assign [14]. Hence, the particular NMR sample was re-checked for purity by radio-HPLC, and indeed, only 12% of activity of desired 24-[24, 25, 26, 27-3 H]epiCS ([3 H]-**2**) was left whereas 88% of radioactivity was found in a broaden peak with higher retention on the column (**Figures 3** and **4**). Nevertheless, authors reported secure procedure to remove chromatographic solvents and formulate the high-SA BRs for application in biochemical experiments. Combined HPLC fractions (each about 2 mL) were first enriched with glycerol (300 mL) (acts as an antioxidant and also prevents risk of getting dryness of labeled samples) and 10 mg of (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (acts as additional antioxidant with no harm in eventual biological experiments) [14]. When methanol and water evaporated on

**Figure 3.** HPLC radiodetector chromatogram of [3 H]-**2** after purification.

**Figure 4.** HPLC radiodetector chromatogram; fast decomposition of [3H]-2 when handled improperly - after its simple evaporation to dryness and storage in DMSO-*d6* overnight.

CentriVap, the residual glycerol solution was further diluted with water (2 mL), the radioactivity was determined and the concentration of 24-[24, 25, 26, 27-3 H]epiCS ([3 H]-**2**) was afterwards adjusted to 1 mCi/mL with a glycerol/water (1:1) mixture. The concentration of Trolox in the final formulation needed to be adjusted to 0.5%. For maximal stability of prepared samples, storage of 1 mCi aliquots in liquid nitrogen is recommended.

## **2.2. BRs with reasonable high SA of tritium**

for 2 h. In one-pot synthesis, derivative **10** was deisopropylinated and deacetylated provided

O2

(1 mL)] cooled to 0°C by an ice bath [14] . The de-isopropylidation of the (22*R*,23*R*,24*R*)-2α,3α-

tritium signals in C-25, C-26, and -27, and is in accordance with the determined SA, indicating

The free BRs with a high SA are extremely sensitive to radiolysis if stored improperly. Authors reported one representative example—when a sample was evaporated to dryness and used

Hence, the particular NMR sample was re-checked for purity by radio-HPLC, and indeed,

radioactivity was found in a broaden peak with higher retention on the column (**Figures 3** and **4**). Nevertheless, authors reported secure procedure to remove chromatographic solvents and formulate the high-SA BRs for application in biochemical experiments. Combined HPLC fractions (each about 2 mL) were first enriched with glycerol (300 mL) (acts as an antioxidant and also prevents risk of getting dryness of labeled samples) and 10 mg of (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (acts as additional antioxidant with no harm in eventual biological experiments) [14]. When methanol and water evaporated on

H]-**2** after purification.

, afterwards full deacetylation was accomplished by methanol solution of CH3

conditions was made possible to obtain 3.5 mCi of pure 24-[24, 25, 26, 27-3

), the signals in 3

H]epicastasterone ([3

H]epicastasterone **11** was carried out by freshly prepared chloroform

H NMR spectra of both [3

H]epiCS ([3

H]-**1**), the Baeyer-Villiger oxidation on fully pro-

(20 ml), trifluoroacetic acid (100 ml), CHCl3

H NMR spectrum were difficult to assign [14].

H]epiBL (**11**) was carried out by wet

H]-**1** and [3

H]-**2**) was left whereas 88% of

H]-**2**) with RCP >97%

H]-epiBL ([3

ONa. This

H]-**2** show

H]-**1**)

by HPLC purification, 40 mCi of 24-[24, 25, 26, 27-3

solution of trifluoroperoxyacetic acid [30% H2

with RCP > 97% and SAMS = 98 Ci/mmol. The 3

*2.1.1. Stability of BRs possessing very high SA*

only 12% of activity of desired 24-[24, 25, 26, 27-3

diacetoxy-22,23-isopropylidenedioxy-24-[24, 25, 26, 27-3

H]epibrassinolide ([3

16 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

and SAMS = 99.4 Ci/mmol [14].

3.4 tritium atoms per molecule.

for the NMR analysis (DMSO-*d6*

**Figure 3.** HPLC radiodetector chromatogram of [3

To get [24, 25, 26, 27-3

FeCl3

tected 24-[24, 25, 26, 27-3

#### *2.2.1. Reductive tritium-dehalogenation of generated chlorocarbonates (6 Ci/mmol)*

To get polyhydroxylated steroid regio- and enantio-specifically labeled on the un-exchangeable position of C-3, a general procedure can be effectively used (**Figure 5**) [17]. A suitable precursor for the introduction of tritium, 3β-chloro-2,3-carbonate derivative, is synthetically affordable by a short-reaction sequence from a 2α,3α-dihydroxy steroid *to be labeled* (**Figure 6**) [18]. Chlorocarbonate undergoes reductive tritium dechlorination catalyzed by the [Pd0]/Et3 N system, providing 28-[3β-3 H]homoCS, 24-[3β-3 H]epiCS, and 24-[3β-3 H]epiBL, respectively, in good yield and with high SA (5.8 Ci/mmol; 0.2 tritium per molecule) (**Figure 5**) [19]. A crucial aspect in the reductive dehalogenation of the chloro derivative is the choice of a solvent that would provide a reasonable yield [17]. The optimized reaction conditions turned out to be PdO/CaCO3 (5%)/Et3 N/chlorocarbonate (2:6:1) dissolved in dry EtOAc for all investigated steroids. The successful 3 H-labeling experiments have proven the stereo selectivity of the reductive dehalogenation and afforded a product with high specific activity (5.8 Ci/mmol).

The synthetic procedure starts with transformation of the vicinal 2α,3α-diols of appropriate BR to α-hydroxy ketone by oxidation with a freshly generated dimethyldioxirane (DMD). Such α-hydroxy ketone moiety proved to be an excellent substrate for high-yield enantiospecific formation of 3β-chloro-2,3-carbonate by a reaction with easy-to-handle triphosgene. The key substrate for reductive dechlorination—3β-chloro-2,3-carbonate—was synthesized by a three-step reaction sequence in an overall yield of 46–55% (**Figure 6**; representative synthesis of 24-[3 H]epiCS). To improve the solubility of the starting steroid *(to be labeled)* in a

**Figure 5.** Tritium Pd-catalyzed reductive dehalogenation; (i) T2 /PdO/CaCO3 /Et3 N; (ii) Fe(III), CH2 Cl2 ; (iii) NaOH, 1,4-dioxane; (iv) H2 O2 /TFA, 0°C, 30 min, r.t., 4 h, CHCl3 .

**Figure 6.** Reaction sequence of the synthesis of 24-[3 H]epiCS; [3 H]-**2**.

non-polar solvent, 2,3- and 22,23-vicinal diols were protected. The isopropylidation of both vicinal diol groups by 2,2-dimethoxypropane (10 eqv.) catalyzed by *p*-toluenesulphonic acid in dry CH2 Cl2 turned out to be an elegant protecting strategy. Full conversion was reached in 30 h and the pure product **4** was isolated. Acetonide **4** was further oxidized at the position of C(3)-OH by freshly synthesized dimethyldioxirane (DMD) [20]. The reaction carried out in CH2 Cl2 overnight at 4°C in dark afforded the desired α-hydroxy ketone **13** accompanied by its regioisomer 3-hydroxy ketone, in a ratio of 10:1 in favor of isomer **13**. In general, α-hydroxy ketone **13** is supposed to be isolated in a higher yield when the oxidation of 2,3-unprotected diols takes place; a selective deprotection of acetonide by Ce(NH4 )2 (NO3 )6 in borate buffer followed by the DMD oxidation of the vicinal 2,3-diol group used to provide a higher overall yield of about 5% [21]. However, in the case of the 28-HCS derivative, a partial deprotectionoxidation reaction sequence afforded a drop of an isolated yield of about 15% compared to direct oxidation of the protected derivative. The 3β-chloro-2,3-carbonate **14** was synthesized by a stereospecific reaction of **13** with triphosgene in dry benzene providing a quantitative yield in 3 h (**Figure 7**).

Recent Developments in a Radio-labeling of Brassinosteroids http://dx.doi.org/10.5772/intechopen.68584 19

**Figure 7.** Mechanism of enantiospecific formation of 3β-chloro-2,3-carbonate derivatives of BR.

non-polar solvent, 2,3- and 22,23-vicinal diols were protected. The isopropylidation of both vicinal diol groups by 2,2-dimethoxypropane (10 eqv.) catalyzed by *p*-toluenesulphonic acid

H]epiCS; [3

30 h and the pure product **4** was isolated. Acetonide **4** was further oxidized at the position of C(3)-OH by freshly synthesized dimethyldioxirane (DMD) [20]. The reaction carried out in

followed by the DMD oxidation of the vicinal 2,3-diol group used to provide a higher overall yield of about 5% [21]. However, in the case of the 28-HCS derivative, a partial deprotectionoxidation reaction sequence afforded a drop of an isolated yield of about 15% compared to direct oxidation of the protected derivative. The 3β-chloro-2,3-carbonate **14** was synthesized by a stereospecific reaction of **13** with triphosgene in dry benzene providing a quantitative

diols takes place; a selective deprotection of acetonide by Ce(NH4

**Figure 5.** Tritium Pd-catalyzed reductive dehalogenation; (i) T2

/TFA, 0°C, 30 min, r.t., 4 h, CHCl3

18 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

.

 overnight at 4°C in dark afforded the desired α-hydroxy ketone **13** accompanied by its regioisomer 3-hydroxy ketone, in a ratio of 10:1 in favor of isomer **13**. In general, α-hydroxy ketone **13** is supposed to be isolated in a higher yield when the oxidation of 2,3-unprotected

H]-**2**.

turned out to be an elegant protecting strategy. Full conversion was reached in

/PdO/CaCO3

/Et3

N; (ii) Fe(III), CH2

Cl2

; (iii) NaOH,

)2 (NO3 )6

in borate buffer

in dry CH2

1,4-dioxane; (iv) H2

O2

CH2 Cl2 Cl2

**Figure 6.** Reaction sequence of the synthesis of 24-[3

yield in 3 h (**Figure 7**).

The catalytic reductive dehalogenation of BR–chlorocarbonates was studied with deuterium in the system of 2 H2 /Pd[0]/Et3 N providing appropriate 3β-deutero-2,3-carbonates with 70–80% deuterium enrichment (based on 1 H NMR) at the C-3 position and with an isolated yield of up to 65% (initially on a cheap pregnane analog available in multigame scale). The best results for deuterium dehalogenation were achieved with the molar ratio of PdO/CaCO3 (5%)/Et3 N/chlorocarbonate being 2:6:1 for every BR derivative at a short period of time (6 h). When the amount of the base was too high, it diminished the yield of [2 H]-labeled ethylene carbonate. Various catalysts such as Pd/C (either 5 or 30%), PdO/BaSO4 (10%) used for reduction yielded lower yield of desired carbonate (15–19%). Authors disclosed very significant solvent effect with an impact on the isolated yield as well as the by-products formation. Briefly, the best results provided EtOAc (dry), giving up to a 65% yield of labeled carbonate with 80% 2 H-enrichment. Other solvents used for reduction provided both low conversion and isolated yield of carbonate (0–19%). Androstane chlorocarbonate employed for reductive dehalogenation under similar condition as for pregnane analog provided analogous results (58% yield, 75% 2 H-enrichment at C-3). Surprisingly, in addition to desired labeled carbonate two by-products were detected, isolated, and afterwards characterized in that experiment—the multi-labeled ketone and the multi-labeled alcohol, both in the yield of 15%. The reaction course toward formation of both by-products was further accelerated while protic solvent (MeOH) was used in the reaction [43% (ketone) and 40% (alcohol)]. The reaction conditions used for the labeling of 24-epiCS were the same as described above for the other two steroids. A full conversion of appropriate chlorocarbonate was obtained after a 6-h reaction [PdO/CaCO3 (5%)/Et3 N/substrate 2:6:1] in dry EtOAc. The isolated yield of labeled carbonate was determined to be 31% [D/H at C-3 = 70:30]. Both the by-products, ketone (20%) and alcohol (13%), were isolated too. The use of DMF as solvent reduced the conversion of chlorocarbonate to 45%, the yield of labeled carbonate down to 19% and the formation of by-products to 11 and 10%, respectively.

As was already mentioned, traces of water (partly synthesized by the reduction of PdO with D2 gas) play a crucial role in the suggested mechanism of by-product formation (**Figure 8**) [17]. The initial oxidative addition of *in situ* generated Pd[0] into the chlorocarbonate C(3)-Cl bond forms an organopalladium compound **16**. Such palladium could be partly substituted by traces of water to form appropriate hydroxyl carbonate **17**, which afterwards undergoes ring opening, leading to ketone **18**. Unsaturated ketone **19** is formed by an elimination of carbonic acid from ketone **18** (Pathway A), which could be *in situ* reduced by a system of D2 /Pd[0] providing 1,2-deuterium-labeled ketone **20** and **21**, respectively. The other discussed pathway (B) involves an elimination of carbon dioxide, affording α-hydroxyketone, and the subsequent

**Figure 8.** Mechanism of multi-labeled by-products formation.

elimination of H2 O leading to **19**. Authors declare a statement that theoretical pathway B was not supported by any analytical findings. First, the α-hydroxyketone was not detected in the reaction mixture. Moreover, the single experiment using synthesized α-hydroxyketone and reaction conditions simulating conditions used for reductive deuterium dehalogenation, yielded ultimately 2α,3β-dihydroxy-3α[2 H] derivative in an isolated 85% yield and no other product was identified in the reaction mixture. In advance, the absolute configuration at C-2 and C-3 of 2α,3β-dihydroxy derivative was confirmed by was confirmed by X-ray analysis affording ORTEP diagram. A regular ESI-MS analysis of **20** and **21** measured in CH3 OH has confirmed the structure of multi-labeled ketones with the distribution of deuterium 1D/2D being 70:30. The second by-product, multi-labeled alcohols **22** and **23**, are most likely formed by a Pd-catalyzed reduction of the ketones **20** and **21** by D2 . The structure of **22** and **23** was confirmed by 1 H NMR as well as ESI-MS with a deuterium distribution 1D/2D/3D being 10:50:40.

In view of the favorable results of deuterium experiments, this protocol was followed using tritium gas. Tritium dehalogenation experiment was designed following the optimized reaction conditions [PdO/CaCO3 (5%)/Et3 N/substrate 2:6:1, dry EtOAc]. 3 H-labeled 24-epiCS and 24-epiBL, was synthesized when the appropriate 3β-chloro-2,3-carbonate **14** was used for a reaction with carrier-free tritium gas (600 mbar), PdO/CaCO3 (5%), and in presence of base Et3 N. The reaction proceeded at 25°C for 17 h (**Figure 6**). The reduction yielded 5.9 mCi of the desired 3 H-labeled carbonate **15**, and two further unidentified by-products (21.3 and 12.4 mCi) were detected. By 1 H NMR was determined a specific activity of **15** at 5.8 Ci/mmol (which accounts for 0.2 tritium atom per molecule). Compared to previous deuterium comprehensive study, the obtained tritium enrichment was lower than expected. Authors speculate that the significant drop in SA was caused by the reduced pressure of tritium gas (600 mbar 3 H2 ) compared to deuterium gas (950 mbar of 2 H2 ) used for the reduction. The only signal in the 3 H NMR spectrum (the singlet at δ 4.8 ppm) explicitly determined the regio- and stereo-specificity of the reduction. The deprotection of the isopropylidene group in the side chain was carried out by wet FeCl3 in CH2 Cl2 . The last step—hydrolysis of 3 H-labeled carbonate **15**—was accomplished by NaOH (0.5 M) in 1,4-dioxane (1:1). Desired [3 H]-**2** (3.8 mCi, 5.8 Ci/mmol) was purified by preparative radio-HPLC. Aliquot of [3 H]-**2** was further employed for Baeyer-Villiger oxidation leading to [3 H]-**1** (0.3 mCi, SA 5.8 Ci/mmol).

The SA of isolated [3 H]-**2** was about four times lower than was predicted based on the previous comprehensive deuterium-using study. To further investigate the influence of a metal catalyst on the SA and the yield of the labeled product, it was considered the use of the Pd[0] catalyst instead of the Pd[II]O catalyst, where the generation of Pd[0] is accompanied by the formation of tritiated water. The suggested mechanism of the by-products formation explained how the traces of water significantly reduce the yield of the labeled product desired (**Figure 8**). Hence, to suppress synthesis of multi-labeled by-products Pd on charcoal was employed at synthesis of 28-[3β-3 H]homocastasterone [19]. The catalyst/base/substrate ratio for the tritium experiment was kept identical to the previous experiment of the tritium labeling of 24-epiCS—2:6:1. Carrier-free tritium released over the reaction mixture (738 mbar, 11.5 Ci, 180 µmol) was left to react for 24 h at room temperature. Then, the analytical radio-HPLC proved the formation of the desired 3 H-labeled carbonate (13.3 mCi) and two other by-products (42.8 and 24.7 mCi) were detected. The SA of 3 H-carbonate was determined by 1 H NMR as 5.8 Ci/mmol (based on the decrease of the corresponding 1 H signal intensity in the labeled position). The deprotection of the 22,23-isopropylidene group by treatment with wet FeCl3 in CH2 Cl2 was completed within 10 min. The crude 3β-tritio-2,3-carbonate was directly hydrolyzed by 0.5 M aqueous NaOH in 1,4-dioxane. The crude product was purified on the semi-prep radio-HPLC, affording 5.3 mCi of [3 H]-(**3**) of SA 5.8 Ci/mmol with radiochemical purity (RCP) >97%. Both by-products were isolated, and 3 H and 1 H NMR measured. The structure of multi-labeled by-products is believed to be similar to those that were recently described (**Figure 9**) for androstane and 24-epiCS (i.e., labeled 24-epityphasterol and 3-dehydro-24-epiteasteron, respectively) derivatives. In this case, these by-products are supposed to be 22,23-isopropylidene-protected multi-labeled [1, 2, 3-3 H]-28-homotyphasterol and [1, 2-3 H]-3-dehydro-28-homoteasterone derivatives (**Figure 9**).

#### *2.2.2. Catalytic reduction of 24-methylene BRs (SA = 2 Ci/mmol)*

elimination of H2

firmed by 1

Et3

3

desired 3

were detected. By 1

tion conditions [PdO/CaCO3

compared to deuterium gas (950 mbar of 2

yielded ultimately 2α,3β-dihydroxy-3α[2

**Figure 8.** Mechanism of multi-labeled by-products formation.

by a Pd-catalyzed reduction of the ketones **20** and **21** by D2

(5%)/Et3

reaction with carrier-free tritium gas (600 mbar), PdO/CaCO3

O leading to **19**. Authors declare a statement that theoretical pathway B was

H] derivative in an isolated 85% yield and no other

. The structure of **22** and **23** was con-

H-labeled 24-epiCS and

(5%), and in presence of base

) used for the reduction. The only signal in the

OH has

H2 )

not supported by any analytical findings. First, the α-hydroxyketone was not detected in the reaction mixture. Moreover, the single experiment using synthesized α-hydroxyketone and reaction conditions simulating conditions used for reductive deuterium dehalogenation,

product was identified in the reaction mixture. In advance, the absolute configuration at C-2 and C-3 of 2α,3β-dihydroxy derivative was confirmed by was confirmed by X-ray analysis

confirmed the structure of multi-labeled ketones with the distribution of deuterium 1D/2D being 70:30. The second by-product, multi-labeled alcohols **22** and **23**, are most likely formed

In view of the favorable results of deuterium experiments, this protocol was followed using tritium gas. Tritium dehalogenation experiment was designed following the optimized reac-

24-epiBL, was synthesized when the appropriate 3β-chloro-2,3-carbonate **14** was used for a

accounts for 0.2 tritium atom per molecule). Compared to previous deuterium comprehensive study, the obtained tritium enrichment was lower than expected. Authors speculate that the significant drop in SA was caused by the reduced pressure of tritium gas (600 mbar 3

H NMR spectrum (the singlet at δ 4.8 ppm) explicitly determined the regio- and stereo-specificity of the reduction. The deprotection of the isopropylidene group in the side chain was

H2

N. The reaction proceeded at 25°C for 17 h (**Figure 6**). The reduction yielded 5.9 mCi of the

H-labeled carbonate **15**, and two further unidentified by-products (21.3 and 12.4 mCi)

H NMR was determined a specific activity of **15** at 5.8 Ci/mmol (which

H NMR as well as ESI-MS with a deuterium distribution 1D/2D/3D being 10:50:40.

N/substrate 2:6:1, dry EtOAc]. 3

affording ORTEP diagram. A regular ESI-MS analysis of **20** and **21** measured in CH3

20 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

An elegant and fast strategy to get BRs labeled by tritium was briefly communicated by Yokota et al. [22]. [3 H]-BRs synthesized on demand at Amersham International (Amersham, UK) were used for comparative analysis in stems and seeds by radioimmunoassay. The labeling strategy was based on platinum-catalyzed reduction of 24-methylene position of available BR (dolichosterone and dolicholide) in carrier-free tritium atmosphere (**Figure 10**). Reduction of dolichosterone afforded two epimers 24-[24, 28-3 H]castasterone (8.1 mCi, SA = 2.2 Ci/mmol) and 24-[24, 28-3 H]epicastasterone [3 H]-(**2**) (6.5 mCi, SA not determined), respectively, which were then separated on HPLC. Analogically, reduction of dolicholide provided 24-[24, 28-3 H] brassinolide (10.6 mCi, SA = 2.3 Ci/mmol) and 24-[24, 28-3 H]epibrassinolide [3 H]-(**1**) (4.0 mCi, SA not determined). Authors stated unexpectedly low SA of gained products (theoretical SA was supposed to be over 40 Ci/mmol). Detailed synthetic procedure and analysis were not reported.

**Figure 9.** Multi-labeled by-products.

22 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

#### **2.3. BRs with very low SA of tritium (10−3 Ci/mmol)**

Tritium-labeled protected 24-epiCS and 24-epiBL [3 H]-**1** were for the first time prepared by Kolbe et al. [23]. By very simple procedure of base-catalyzed exchange reaction with tritiated water (SA = 20.10−3 Ci/mmol—accounts for 0.7 × 10−3 tritium atom per molecule HTO) to the enolizable alpha positions of C-6 ketone of tetracetate 24-epiCS **27** (**Figure 11**). By this method, it is theoretically possible to exchange hydrogen on three distinctive positions (C-5α, C-7α, and C-7β). However, the obtained SA of BRs was very low (6.10−3 Ci/mmol), about 3-4 orders lower than that provided by methods described above. Because of low SA of such prepared material cannot be used in receptor studies at all. The other drawback of this procedure is chemical exchangeability of labels, which inevitably leads to loss of label in protic solvents (water) during biological experiments and disqualifies this approach from use in ADME studies. Three-step reaction sequence provided 24-[5, 7, 7-3 H]epiBL with SA 6.10−3 Ci/mmol; labeling step leading to protected 24-[3 H]epiCS **27**, followed by a Baeyer-Villiger oxidation with CF3 COOOH and sequence was accomplished by hydroxy group deprotection [20] . The same sequence was also performed with 2,3,22,23-bis-isopropylidenedioxy-24-epiCS **29** as starting compound. The advantage of the use of isopropylidene protecting groups over acetate group is the deprotection step. The lactone ring is stable under acidic hydrolysis conditions used for isopropylidene deprotection, which occurs simultaneously with Baeyer-Villiger oxidation. The basic hydrolysis needed for the acetate group cleavage leads to lactone hydrolysis and acid catalyzed re-lactonization is needed (**Figure 11**).

A base-catalyzed exchange was used for labeling of biogenetic brassinosteroids precursors [24]. 24-[5, 7, 7-3 H]epiteasterone (SA = 1.5 10−3 Ci/mmol), 6-oxo-24β-methyl-22-dehydro[5, 7, 7-3 H]cholestenol (SA = not indicated), and 6-oxo-24-[5, 7, 7-3 H]epicampestanol (SA = 3.5 10−3 Ci/mmol ), respectively, were partly labeled on positions of C-5 and C-7 by reaction in sealed

**Figure 11.** Low SA possessing 24-[3 H]-epiBL.

**Figure 10.** Metal-catalyzed 24-methylene BR tritiation.

**Figure 9.** Multi-labeled by-products.

22 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

ampoule with tritiated water (SA = 14.10−3 Ci/mmol—accounts for 0.5 × 10−3 tritium atom per molecule HTO) in presence of base Et3 N (**Figure 12**). As mentioned above this simple methodology affords poor SA (in order of 10−3 Ci/mmol) of BRs labeled on an exchangeable positions thus prone to loss of label later on. On the other hand, this approach provides high yield (>45%) of desired material.

**Figure 12.** 24-[3 H]epiteasterone and 6-oxo-24-[3 H]epicampestanol.

#### **2.4. BRs labeled by carbon-14 (SA = 56.8 × 10−3 Ci/mmol)**

Till now, only one report on 14C-labeled brassinosteroids is available in the literature. In 1989, Seo et al. described the synthesis of [14C]-labeled (22*R*,23*R*)- and (22*S*,23*S*)-epiCS **50** and **51**, and epiBL **52** and **53**, respectively [25]. The obtained BRs possessed top SA (56.8 × 10−3 Ci/mmol) possible to reach while doing labeling by carbon-14. Because of multi-step synthetic sequence (>10 steps) needed, the reported overall yield of labeled products is 3.2 and 4.5%. The C-4 position in 24-epiBL **52** was selected for [14C]-labeling because of its stability to metabolic loss and easy way to do the preparation. According to the well-established method for 14C incorporation into the C-4 position of steroids, the enol lactone **41** was synthesized from the starting material brassicasterol **36** in five steps (**Figure 13**). Bridged ketone **42** was prepared from lactone **41** after its treatment with [14C]methyl iodide. Alkaline treatment of **42** provided [4-14C] enone **43**. Gentle acid catalyzed acetylation of enone **43** with isopropenyl acetate led to the enol acetate **44** that was afterwards reduced by NaBH4 to give [4-14C]brassicasterol **45**. Simple mesylation of **45** afforded **46** which was treated with sodium carbonate providing the major product 3,5-cyclo-6-ol **47** isolated in 92% yield (**Figure 14**). Jones oxidation of **47** then gave 3,5-cyclo-6-one **48**. Rearrangement of **48** moderated by lithium bromide and camphor sulfonic acid in dimethyl acetamide afforded 2,22-diene-6-one **49** in quantitative yield. Oxidation of **49** with osmium tetroxide led to a stereo isomeric mixture of 2,3,22,23-tetraols–24-(22*R*,23*R*)- [14C]epiCS **50** and 24-(22*S*,23*S*)-[14C]epiCS **51** which were separated. The final Baeyer-Villiger oxidation of ketone C-6 with TFA in dichloromethane afforded the (22*R*,23*R*)-7-oxa-lactone **52** accompanied with its 6-oxa isomer **56** as a minor product. Analogically, (22*S*,23*S*)-tetraol provided the (22*S*,23*S*)-7-oxa-lactone **53** accompanied with its 6-oxa isomer **57**. The ultimate products (22*R*,23*R*)-24-[14C]epiBL **52** and (22*S*,23*S*)-24-[14C]epiBL **53** were isolated in overall 3.20 and 4.46% radiochemical yield (toward Ba14CO3 used as an initial source or radio-label), respectively. SA of prepared BRs was 56.8 × 10−3 Ci/mmol.

ampoule with tritiated water (SA = 14.10−3 Ci/mmol—accounts for 0.5 × 10−3 tritium atom per

24 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

odology affords poor SA (in order of 10−3 Ci/mmol) of BRs labeled on an exchangeable positions thus prone to loss of label later on. On the other hand, this approach provides high yield

Till now, only one report on 14C-labeled brassinosteroids is available in the literature. In 1989, Seo et al. described the synthesis of [14C]-labeled (22*R*,23*R*)- and (22*S*,23*S*)-epiCS **50** and **51**, and epiBL **52** and **53**, respectively [25]. The obtained BRs possessed top SA (56.8 × 10−3 Ci/mmol) possible to reach while doing labeling by carbon-14. Because of multi-step synthetic sequence (>10 steps) needed, the reported overall yield of labeled products is 3.2 and 4.5%. The C-4 position in 24-epiBL **52** was selected for [14C]-labeling because of its stability to metabolic loss and easy way to do the preparation. According to the well-established method for 14C incorporation into the C-4 position of steroids, the enol lactone **41** was synthesized from the starting material brassicasterol **36** in five steps (**Figure 13**). Bridged ketone **42** was prepared from lactone **41** after its treatment with [14C]methyl iodide. Alkaline treatment of **42** provided [4-14C] enone **43**. Gentle acid catalyzed acetylation of enone **43** with isopropenyl acetate led to the

H]epicampestanol.

mesylation of **45** afforded **46** which was treated with sodium carbonate providing the major product 3,5-cyclo-6-ol **47** isolated in 92% yield (**Figure 14**). Jones oxidation of **47** then gave 3,5-cyclo-6-one **48**. Rearrangement of **48** moderated by lithium bromide and camphor sulfonic acid in dimethyl acetamide afforded 2,22-diene-6-one **49** in quantitative yield. Oxidation of

N (**Figure 12**). As mentioned above this simple meth-

to give [4-14C]brassicasterol **45**. Simple

molecule HTO) in presence of base Et3

**2.4. BRs labeled by carbon-14 (SA = 56.8 × 10−3 Ci/mmol)**

H]epiteasterone and 6-oxo-24-[3

enol acetate **44** that was afterwards reduced by NaBH4

(>45%) of desired material.

**Figure 12.** 24-[3

**Figure 13.** Synthetic pathway to 24-[14C]epiCS and 24-[14C]epiBL.

26 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

**Figure 14.** Synthetic pathway to 24-[14C]epiCS and 24-[14C]epiBL.


**3. Test Results and Analysis**

### **3. Test Results and Analysis**  *3.1. Determination of Compressive Strength*

*3.1. Determination of Compressive Strength*  The cubic compressive strength of CSG material was followed the Test Rules for Hy-The cubic compressive strength of CSG material was followed the Test Rules for Hydraulic Concrete (SL352-2006).

draulic Concrete (SL352-2006). In this test, four specimens were prepared as one group. After testing, the largest discrete data were removed and the other three groups were averaged. The test results In this test, four specimens were prepared as one group. After testing, the largest discrete data were removed and the other three groups were averaged. The test results were in accordance with the Rules.

### were in accordance with the Rules. *3.2. Analysis of the Test Data*

### *3.2. Analysis of the Test Data*  3.2.1. Effect of Fly Ash Content on the Compressive Strength of CSG Material

3.2.1. Effect of Fly Ash Content on the Compressive Strength of CSG Material Fly ash is a type of artificial pozzolanic material made of silica or silica-alumina. It possesses minimal or no cementing value. However, in the presence of water, fly ash pow-Fly ash is a type of artificial pozzolanic material made of silica or silica-alumina. It possesses minimal or no cementing value. However, in the presence of water, fly ash powder will react with Ca(OH)2 at room temperature to form a compound with cementing properties. The pozzolanic activity of fly ash is reflected by its cementing.

der will react with Ca(OH)2 at room temperature to form a compound with cementing properties. The pozzolanic activity of fly ash is reflected by its cementing. The test results are shown in Figure 2 for the analysis of the relationship between age, fly ash content, and strength.

The test results are shown in Figure 2 for the analysis of the relationship between age, fly ash content, and strength. As detailed in Figure 2, the rules can be found as follows: (1) the compressive strength of CSG increases with age. The compressive strength of CSG at 90 d is generally 10–30% higher than that at 28 d. (2) When the cement content is 50 kg/m3 and 60 kg/m3 , respectively, the compressive strength of CSG increases with the addition of fly ash content. When the cement content is 50 kg/m3 , the increase of compressive strength at 28 d is greater than that at 90 d. When the cement content is 60 kg/m3 , the increase of compressive strength at 90 d is greater than that at 28 d [35].

**Figure 2.** The relationship between the compressive strength of CSG material and the age / the content of fly ash. **Figure 2.** The relationship between the compressive strength of CSG material and the age/the content of fly ash.

As detailed in Figure 2, the rules can be found as follows: (1) the compressive strength of CSG increases with age. The compressive strength of CSG at 90 d is generally 10–30% higher than that at 28 d. (2) When the cement content is 50 kg/m3 and 60 kg/m3, respectively, the compressive strength of CSG increases with the addition of fly ash content. When the cement content is 50 kg/m3, the increase of compressive strength at 28 d is greater than that at 90 d. When the cement content is 60 kg/m3, the increase of compressive strength at 90 d is greater than that at 28 d [35]. The test results can be explained with two-phase hydration reaction of the fly ashcement system. First, the induction stage, during which the soluble ions on the surface of fly ash particles dissolve, which affects the nucleation of Ca(OH)2 and C-S-H hydrates and retards the initial level of C3A in cement. Meanwhile, the hydration of cement is delayed, as the fine size of fly ash particles leads to their easy adhesion to the surface of cement particles under physical action. In addition, Ca(OH)2 and C-S-H produced by cement hydration wrap the surface of fly ash and prevent the hydration of fly ash particles. Therefore, the fly ash with early age has low self-activity. Second, the acceleration stage, during which Ca(OH)2 and C-S-H begin to nucleate and grow. Fly ash particles provide additional accommodation for the precipitation of C-S phase hydrates in cement through many new surfaces. In this way, fewer C-S phase hydrates will precipitate on the surface of cement particles, and the dissolution of C3S will accelerate. As a result, the hydration of cement is promoted. Therefore, during the second phase, the presence of fly ash facilitates The test results can be explained with two-phase hydration reaction of the fly ashcement system. First, the induction stage, during which the soluble ions on the surface of fly ash particles dissolve, which affects the nucleation of Ca(OH)2 and C-S-H hydrates and retards the initial level of C3A in cement. Meanwhile, the hydration of cement is delayed, as the fine size of fly ash particles leads to their easy adhesion to the surface of cement particles under physical action. In addition, Ca(OH)2 and C-S-H produced by cement hydration wrap the surface of fly ash and prevent the hydration of fly ash particles. Therefore, the fly ash with early age has low self-activity. Second, the acceleration stage, during which Ca(OH)2 and C-S-H begin to nucleate and grow. Fly ash particles provide additional accommodation for the precipitation of C-S phase hydrates in cement through many new surfaces. In this way, fewer C-S phase hydrates will precipitate on the surface of cement particles, and the dissolution of C3S will accelerate. As a result, the hydration of cement is promoted. Therefore, during the second phase, the presence of fly ash facilitates the hydration of cement. Moreover, due to the nucleation of cement hydration products, the permeability of fly ash coating increases and leads to faster hydration of the fly ash. As fly as hydration consumes more Ca(OH)2, the hydration of cement is promoted further. During this stage, cement and fly ash promote and accelerate each other's hydration at the same time. This phenomenon become obvious with the development of age, which is highlighted by the significant increase of strength of CSG material at the later stage. Therefore, considering the gradual development of material strength, it is appropriate to choose 90 d or 180 d strength as the design strength of CSG dam.

### the hydration of cement. Moreover, due to the nucleation of cement hydration products, the permeability of fly ash coating increases and leads to faster hydration of the fly ash. 3.2.2. Optimal Fly Ash Content of CSG Material

As fly as hydration consumes more Ca(OH)2, the hydration of cement is promoted further. During this stage, cement and fly ash promote and accelerate each other's hydration at the same time. This phenomenon become obvious with the development of age, which is highlighted by the significant increase of strength of CSG material at the later stage. Therefore, considering the gradual development of material strength, it is appropriate to choose Most hydraulic projects will add fly ash to cementitious materials, and the RCC dam has the highest proportion of fly ash addition (70%) (Jiangya Hydropower Station). Technical Guidelines for Damming with Cemented Granular Materials (SL678-2014) also proposes including fly ash into cement gravel and sand material for dam construction, but without mentioning the most optimal and economical fly ash content.

90 d or 180 d strength as the design strength of CSG dam. 3.2.2. Optimal Fly Ash Content of CSG Material Most hydraulic projects will add fly ash to cementitious materials, and the RCC dam has the highest proportion of fly ash addition (70%) (Jiangya Hydropower Station). Technical Guidelines for Damming with Cemented Granular Materials (SL678-2014) also proposes including fly ash into cement gravel and sand material for dam construction, but During the test, prepared the cement content of 50 kg/m3 and 60 kg/m3 , added fly ash of 20 kg/m3 , 30 kg/m3 , 40 kg/m3 , 50 kg/m3 , 60 kg/m3 , 80 kg/m3 , or 100 kg/m3 , respectively, into the cement and waited for 90 d maintenance to make CSG materials to see the difference of their compressive strengths, with results drawn in the figure below. According to the figure, as the fly ash content increases, the compressive strength reaches a peak before falling down to a level. The CSG material test proves that an optimal content of fly ash could be found. The compressive strength of materials will reach its maximum

without mentioning the most optimal and economical fly ash content.

*Crystals* **2021**, *11*, x FOR PEER REVIEW 6 of 10

when both cement and fly ash contents stand at 50 kg/m3 or 60 kg/m3 , as highlighted in Figure 3. falling down to a level. The CSG material test proves that an optimal content of fly ash could be found. The compressive strength of materials will reach its maximum when both cement and fly ash contents stand at 50 kg/m3 or 60 kg/m3, as highlighted in Figure 3.

During the test, prepared the cement content of 50 kg/m3 and 60 kg/m3, added fly ash of 20 kg/m3, 30 kg/m3, 40 kg/m3, 50 kg/m3, 60 kg/m3, 80 kg/m3, or 100 kg/m3, respectively, into the cement and waited for 90 d maintenance to make CSG materials to see the difference of their compressive strengths, with results drawn in the figure below. According to the figure, as the fly ash content increases, the compressive strength reaches a peak before

**Figure 3.** Optimal content of fly ash of CSG material. **Figure 3.** Optimal content of fly ash of CSG material.

According to our analysis, as the design of mixed proportions in this test is based on previous research results, the total water consumption of the mixture is fixed by selecting a water–binder ratio of 1.0 and sand ratio of 20%. [35,36] At the initial stage, the strength of CSG material increases with the increase of fly ash content and the water amount is enough to support the hydration of cement and fly ash. The increase of fly ash facilitates C-S-H cementing; when all the water is consumed during the hydration of cement and fly ash, the fly ash added will only serve as a filler, which fill in the concrete voids or attach to the surface of cement particles. At this stage, more fly ash will affect the formation of Ca(OH)2, C-S-H, which leads to the declining strength of CSG materials. Therefore, there is an optimal fly ash content in CSG material, and it is defined to be 50% of the contentious material (cement + fly ash). According to our analysis, as the design of mixed proportions in this test is based on previous research results, the total water consumption of the mixture is fixed by selecting a water–binder ratio of 1.0 and sand ratio of 20%. [35,36] At the initial stage, the strength of CSG material increases with the increase of fly ash content and the water amount is enough to support the hydration of cement and fly ash. The increase of fly ash facilitates C-S-H cementing; when all the water is consumed during the hydration of cement and fly ash, the fly ash added will only serve as a filler, which fill in the concrete voids or attach to the surface of cement particles. At this stage, more fly ash will affect the formation of Ca(OH)2, C-S-H, which leads to the declining strength of CSG materials. Therefore, there is an optimal fly ash content in CSG material, and it is defined to be 50% of the contentious material (cement + fly ash).

### **4. Numerical Regression Analysis 4. Numerical Regression Analysis**

Regression analysis is an important branch of mathematical statistics and an important statistical tool to study the correlation between variables. It is well applied in Regression analysis is an important branch of mathematical statistics and an important statistical tool to study the correlation between variables. It is well applied in many areas, such as seeking empirical formulas or establishing mathematical models.

many areas, such as seeking empirical formulas or establishing mathematical models. According to the test, fly ash content has significant influence on the compressive strength of CSG material. Based on the theory of linear regression model, Formula (1) is built to reflect the statistical relationship between 90 d compressive strength and fly ash content in CSG material: According to the test, fly ash content has significant influence on the compressive strength of CSG material. Based on the theory of linear regression model, Formula (1) is built to reflect the statistical relationship between 90 d compressive strength and fly ash content in CSG material:

$$X\_F = a\_f x\_f^2 + b\_f x\_f + c\_f \tag{1}$$

2 *XF ff ff f* = ++ *ax bx c* (1) where *a f* , *bf* , and *c f* are statistical constants.

where *f a* , *f b* , and *f c* are statistical constants. After calculation, the regression equation parameters of fly ash content and compressive strength are obtained and shown in Table 4.

After calculation, the regression equation parameters of fly ash content and compressive strength are obtained and shown in Table 4. As illustrated in Table 4, the fitting correlation coefficients are greater than 0.90 and the standard errors are less than 0.58 in all cases. The fitting strength of each regression As illustrated in Table 4, the fitting correlation coefficients are greater than 0.90 and the standard errors are less than 0.58 in all cases. The fitting strength of each regression equation and measured values have demonstrated excellent correlation with regard to 90 d compressive strength of CSG material, with small deviation of regression values and measured values, and high fitting accuracy. The comprehensive analysis shows that the test results suit the mathematical model of linear regression.

Through numerical analysis, the maximum value of compressive strength under different fly ash content can be obtained, where x represents the optimal fly ash content and y represents the maximum compressive strength of CSG material, as detailed in Table 4.

The numerical analysis shows that when (1) 50 kg/m3 of cement content is combined with 51 kg/m3 of fly ash content, or (2) the cement and fly ash contents are 60 kg/m3 and 59 kg/m3 , respectively, the compressive strength of CSG material is the highest in 90 d. Therefore, when fly ash content: cement content ≈ 1:1 or when the fly ash content is 50% of cementitious materials (cement + fly ash), the "optimal content" is achieved. The "optimal fly ash content" of CSG material was verified again through numerical analysis.

**Table 4.** Regression analysis parameter table.